Methods for modifying human antibodies by glycan engineering

ABSTRACT

Methods for making modified Fc regions of antibodies and antibody fragments, both human and humanized, and having enhanced stability and efficacy, are provided. Antibodies comprising Fc regions with core fucose residues removed, and attached to oligosaccharides comprising terminal sialyl residues, are provided. Antibodies comprising homogeneous glycosylation of Fc regions with specific oligosaccharides are provided. Fc regions conjugated with homogeneous glycoforms of monosaccharides and trisaccharides, are provided. Methods of preparing human antibodies with modified Fc using glycan engineering, are provided.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This patent application claims the benefit or priority of, and is a Continuation-in-Part of, U.S. application Ser. No. 12/959,351, filed Dec. 2, 2010, now U.S. Pat. No. 10,087,236, which claims priority of U.S. Provisional Patent Application Ser. No. 61/265,757, filed Dec. 2, 2009. This patent application is also a Continuation-in-Part of U.S. application Ser. No. 16/018,400, filed Jun. 26, 2018, which claims priority to U.S. application Ser. No. 14/723,297, filed May 27, 2015, now U.S. Pat. No. 10,023,892, which claims priority to US provisional applications U.S. Ser. No. 62/003,136, filed May 27, 2014, U.S. Ser. No. 62/003,104, filed May 27, 2014 U.S. Ser. No. 62/003,908, filed May 28, 2014, U.S. Ser. No. 62/020,199, filed Jul. 2, 2014, and U.S. Ser. No. 62/110,338, filed Jan. 30, 2015, the contents of each of which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 19, 2019, is named G2112-00604_SL.txt and is 6,550 bytes in size.

TECHNICAL FIELD OF THE INVENTION

This invention relates to the field of antibodies. In particular, the application relates to methods for modifying glycosylation states of human monoclonal antibodies (mAbs) by glycan engineering. More particularly, the application relates to uniformly glycosylated glycoforms of Fc regions of human mAbs and methods for preparing the antibodies by glycan engineering.

BACKGROUND OF THE INVENTION

Antibodies, also known as immunoglobulins (Ig), are glycoproteins that play a central role in immune responses. IgG antibodies (Abs) and fragments of IgG Abs have become major biotherapeutics for treating human diseases.

There are five functional classes of immunoglobulins, i.e., immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin E (IgE). Among them, IgG is the most abundant immunoglobulin in serum.

The variable region (Fab) of the antibody molecule is involved in direct binding of the target antigen. All naturally-occurring antibodies each include a constant domain known as the Fc (Fragment, crystallizable) region, which is composed of constant domains depending on the class of the antibody. By binding to specific proteins the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils. (Woof J, Burton D (2004) Nat Rev Immunol 4 (2): 89-99; Heyman B (1996) Immunol. Lett. 54 (2-3): 195-199). Glycosylation of the antibody Fc fragment is essential for Fc receptor-mediated activity. (Peipp M. et al., Blood (2008) 112(6):2390-2399).

The constant (Fc) domain of naturally-occurring antibodies is usually N-glycosylated. The linkage of carbohydrates to proteins occurs through N-linked glycosylation—the attachment of a sugar to the amide nitrogen atom on the side chain of asparagines. The linkage to amino acids is generally embedded in a conserved sequence of amino acids. Glycosylation in the Fc region significantly affects Fc effector functions such as activation of complement (Roos et al., J Immunol. (12):7052-7059.2001) or binding to receptors via their constant (invariable sequence) domains (Mimura et al., J Biol. Chem. 2001 Dec. 7; 276(49):45539-45547; Shields et al., J Biol. Chem. 276(9):6591-6604 (2001)).

It has been found that the sequences of the oligosaccharides attached to Fc regions are essential to the stability and function of the antibodies. For example, the addition of sialic acid (SA), sialylation, at Asn 297 on the Fc domain and terminal galactosylation modify the anti-inflammatory properties of immunoglobulins and play a role in rheumatoid arthritis (RA). (Kaneko, Y et al., Science. (2006) 313(5787):670-673).

Human antibodies prepared by conventional methods, such as genetic engineering, comprise mixtures of different glycoforms, i.e., containing different oligosaccharides attached to their Fc regions.

The clinical successes of the antibodies rituximab and alemtuzumab for hematological malignancies, and trastuzumab for solid breast tumors, have demonstrated that human or humanized monoclonal antibodies can be useful drugs for the treatment of cancer. All of these antibodies have been approved and are currently in the market. Yet, a need remains for improving this kind of drugs. For instance, rituximab induces a response only in approximately 60% of the patients with relapsed/refractory low-grade non-Hodgkin's lymphoma.

The preferred type of monoclonal antibody for target-cell killing applications is an IgG1-type mAb, where at least the constant (or the Fc) region is of human origin (i.e., chimeric, humanized, or fully-human IgG1s). IgG1 mAbs can exert their therapeutic effects via three classes of mechanisms. Direct binding of the antibody via its variable region to the target molecule on the surface of cancer cells can lead to cell death or inhibition of tumor growth, for example by triggering apoptosis upon cross-linking of target molecules by the antibody. The Fc region operates in the other two-types of mechanisms: complement-mediated cytotoxicity, and Fcγ receptor (FcγR)-dependent effector functions. Some Fc effector functions of IgG1s are mediated by the interactions of the Fc region of target-cell bound antibody molecules and Fcγ receptors on the surface of immune cells. The Fc domains of immunoglobulins have been shown to have effector functions which are primarily complement fixation and Fc receptor (FcR) binding. appropriate glycosylation at the conserved glycosylation site (N297) of the Fc domain is essential for the efficient interactions between mAbs and Fc receptors (FcR) and for the FcR-mediated effector functions, including antibody-dependent cell mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Fc receptors bind to the constant domains of immunoglobulins and a number of receptors have been defined that are thought to mediate accessory functions including opsonization and ADCC (Daeron, M. Annual Review Immunology, (1997) 15; 203-234, Ravetch and Clynes, Annual Review of Immunology. 1998. 16:421-432).

The conserved glycosylation site on the Fc region of antibodies is a target for modulation of antibody effector functions. The crystal structure of a biosynthetic intermediate of human IgG1, bearing immature oligomannose-type glycans and reported to display increased antibody-dependent cellular cytotoxicity, has shown that glycan engineering can bias the Fc to an open conformation primed for receptor binding. (Crispin M., et al., J. Mol. Biol. 387(5):1061-1066 (2009).)

More specifically, certain glycoforms of a human antibody exhibit improved therapeutic effects while others possess undesired properties. For example, de-fucosylated but glycosylated Herceptin® is at least 50-fold more active in the efficacy of Fcγ receptor IIIa mediated ADCC than those with alpha-1,6-linked fucose residues (Shields, R L, et al. J. Biol. Chem., Vol. 277(30):26733-26740, (2002)). Similar results were reported for Rituximab® and other mAbs (Shinkawa T, et al., J. Biol. Chem. (2003) 278:3466-3473).

Strome et al., (WO 2007/146847) disclose a method of preparing antibodies with homogeneous glycosylation states by first deglycosylating an antibody or antibody fragment and then attaching a chemically synthesized sugar to the protein. This method lacks the ability to control the glycosylation process and does not enable control over the conjugation of specific carbohydrates at specific sites during the formation of a desired glycoform. Thus, there is a need for methods of preparing human antibodies comprising desired glycoform(s) wherein the glycosylation is performed in a stepwise manner so as to improve their therapeutic efficacy.

SUMMARY OF THE INVENTION

Provided herein are methods for engineering the glycan on the Fc region of a human, chimeric, or humanized glycosylated antibody, wherein the glycosylated antibody comprises an oligosaccharide attached at Asn-297, wherein the oligosaccharide includes or excludes a core fucose. In some embodiments the method can include the steps of contacting the antibody with a β-1,4-galactosyltransferase and UDP galactose thereby coupling the terminal unit of the oligosaccharide to the galactose to form an oligosaccharide having a terminal galactose unit, wherein the oligosaccharide having a terminal galactose unit is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody; and contacting the antibody comprising the oligosaccharide having a terminal galactose with an alpha-2,6-sialyltransferase and CMP-Neu5Ac, thereby adding a terminal Neu5Ac to each terminal galactose unit to form an oligosaccharide having a terminal Neu5Ac2Gal2, wherein the oligosaccharide having a terminal Neu5Ac2Gal2 is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody. In some embodiments, the method can include the step of providing the human, chimeric, or humanized glycosylated antibody, wherein the glycosylated antibody. The human, chimeric, or humanized glycosylated antibody can be an IgG, IgE. IgA, IgD, or and IgM. In some embodiments the IgG can be an IgG1, an IgG2, an IgG3, or an IgG4. The human, chimeric, or humanized glycosylated antibody can be a therapeutic antibody selected from the group consist of ravulizumab, sacituzumab, risankizumab, emapalumab, cemiplimab, galcanezumab, fremanezumab, romosozumab, moxetumomab, caplacizumab, lanadelumab, mogamuizumab, erenumab, tildrakizumab, ibalizumab, burosumab, durvalumab, emicizumab, benralizumab, ocrelizumab, guselkumab, inotuzumab, sarilumab, dupilumab, avelumab, brodalumab, atezolizumab, bezlotoxumab, olaratumab, reslizumab, obiltoxaximab, ixekizumab, daratumumab, elotuzumab, necitumumab, idarucizumab, alirocumab, mepolizumab, evolocumab, dinutuximab, secukinumab, nivolumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab, raxibacumab, pertuzumab, brentuximab, belimumab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certolizumab, catumaxomab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab, adalimumab, alemtuzumab, gemtuzumab, trastuzumab, infliximab, palivizumab, basiliximab, daclizumab, rituximab, abciximab, edrecolomab, nebacumab, and muromonab. In some embodiments, the oligosaccharide attached at Asn-297 can include an oligosaccharide moiety having the structure of Gal₂GlcNAc₂Man₃GlcNAc₂, GalGlcNAc₂Man₃GlcNAc₂, or GlcNAc₂Man₃GlcNAc₂ the glycan engineered human, chimeric or humanized antibody can have an Neu5Ac₂Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 in the Fc region. In some embodiments, the method can include a set of CMP-Neu5Ac regeneration enzymes comprising a pyrophosphatase and a cytidine monophosphate kinase, and wherein the CMP-Neu5Ac is regenerated following the addition of the terminal Neu5Ac. The glycan engineered human, chimeric or humanized antibody can be further purified.

Also provided are methods for making an essentially pure population of glycoengineered human, chimeric, or humanized glycosylated antibodies from a population of precursor human, chimeric, or humanized glycosylated antibodies, wherein the precursor glycosylated antibodies have an oligosaccharide attached at Asn-297, wherein the oligosaccharide includes or excludes a core fucose, the method comprising contacting the precursor antibodies with a β-1,4-galactosyltransferase and UDP galactose thereby coupling the terminal unit of the oligosaccharide to the galactose to form an oligosaccharide having a terminal galactose unit, wherein the oligosaccharide having a terminal galactose unit is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody; and contacting the antibodies comprising the oligosaccharide having a terminal galactose with an alpha-2,6-sialyltransferase and CMP-Neu5Ac, thereby adding a terminal Neu5Ac to each terminal galactose unit to form an oligosaccharide having a terminal Neu5Ac2Gal2, wherein the oligosaccharide having a terminal Neu5Ac2Gal2 is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibodies, and wherein the essentially pure population of glycoengineered human, chimeric, or humanized glycosylated antibodies comprises at least about 85% by weight of the glycoengineered human, chimeric, or humanized glycosylated antibodies.

Also provided are pharmaceutical formulations comprising an antibody prepared by the methods disclosed herein. The antibody can have the glycan structure of: Sia₂(a2-6)Gal₂GlcNAc₂Man₃GlcNAc₂ attached to the Asn297 of the Fc region.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawing and detailed description of several embodiments, and also from the appended claims.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows a schematic illustration depicting a pathway (Path A) for modifying a representative human antibody by glycan engineering, using alpha-fucosidase, alpha-2,6-sialyltransferase, and optionally β-1,4-galactosyltransferase.

FIG. 2 shows a schematic illustration depicting a chemoenzymatic pathway (Path B) for preparing human antibodies in a single glycoform by glycan engineering, using various exo-glycosidases, endo-glycosidases, and glycosyltransferases.

FIG. 3 shows a schematic illustration depicting an enzymatic pathway (Path C) for preparing human antibodies in a single glycoform by glycan engineering, using various exo-glycosidases and glycosyltransferases.

FIG. 4 shows a schematic illustration depicting the regeneration of the cofactor CMP-Neu5Ac and UDP-galactose.

FIG. 5A shows a schematic illustration of digestion of heterogeneous glycoforms of Humira® obtained from Chinese hamster ovary (CHO) cells with Endo F2 and Endo F3. The results are shown in Coomassie stained polyacrylamide gels in FIG. 5B.

FIG. 6 shows a schematic illustration of depicting a method of engineering a glycan on the FC region of a glycoantibody.

FIG. 7 is a table listing representative therapeutic antibodies. (Note: Related Antibod fragments and ADCs are shown)

FIG. 8 depicts the results of treatment of Rituximab with β-1,4-galactosyltransferase and UDP galactose.

FIG. 9 is a table showing the amino acid sequences of the heavy chain (SEQ ID NO. 1) of Rituximab and the light chain (SEQ ID NO. 2) of Rituximab.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are modified human antibodies prepared via glycan engineering. The invention relates to the Fc region of an antibody molecule, wherein the Fc region is specifically glycosylated with oligosaccharides that increase the efficacy and stability of the Fc region, and the antibody or antibody fragment comprising the Fc region. In some embodiments the specifically glycosylated Fc fragment comprises a monoclonal antibody, preferably a human or humanized monoclonal antibody. Methods for generating such Fc glycosylated antibodies or antibody fragments by glycan engineering are disclosed herein.

Provided herein are materials and methods for efficient production of homogeneous populations of glycoengineered antibodies having the structure Neu5Ac₂Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 in the Fc region. Such antibodies provide enhanced therapeutic benefits relative to parental antibodies that have heterogeneous glycan structures attached at Asn 297 in the Fc region. Specifically, glycoengineered antibodies have enhanced efficacy of effector cell function mediated via increased FcγRIIIA binding and antibody-dependent cytotoxicity (ADCC) compared to the parental antibodies. The inventors have found that enzymatic treatment of populations of antibodies having heterogeneous glycan structures attached at Asn 297 permitted the in situ conversion of heterogeneous glycan structures to homogeneous glycan structures without the need for removal of the heterogeneous glycan structures followed by their replacement with homogeneous glycan structures.

In one embodiment, a two-step method is provided. In the first step, the native glycoantibody can be treated with β-1,4-galactosyltransferase and UDP galactose. The β-1,4-galactosyltransferase specifically catalyzes the addition of galactose to any glycan structures having a terminal GlcNAc. Exemplary glycan substrates include GalGlcNAc₂Man₃GlcNAc (“G1”), and the GlcNAc₂Man₃GlcNAc (“G0”). The glycan substrates can include or exclude a core fucose, that is, the exemplary structures “G0F” and“G0” respectively. The β-1,4-galactosyltransferase treatment generates an “intermediate” population of antibodies having an oligosaccharide structure having a terminal galactose unit, wherein the oligosaccharide having a terminal galactose unit is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody. An exemplary glycan structure intermediate population antibodies can be Gal₂GlcNAc₂Man₃GlcNAc₂ (G2/G2F) attached at each Asn 297 of the Fc region. For the second step, the “intermediate” population of antibodies can be treated with alpha-2,6-sialyltransferase and CMP-Neu5Ac (also referred to as CMP-sialic acid). The alpha-2,6-sialyltransferase specifically catalyzes the addition of Neu5Ac to the terminal galactose unit of the intermediate population of antibodies produced in the first step as well as to any native antibodies having a terminal galactose unit, to form an oligosaccharide having a terminal Neu5Ac2Gal₂ attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody. Thus, the second step of the method can produce a population of homogeneously glycosylated antibodies having, for example, the structure Neu5Ac₂Gal₂GlcNAc₂Man₃GlcNAc₂ (“G2S2/G2S2F”) attached at each Asn 297 in the Fc region.

The methods disclosed herein can be applied to a glycosylated antibody, for example, a human, chimeric, or humanized glycosylated antibody. The glycosylated antibody can include or exclude a core fucose. The glycosylated antibody can be a therapeutic antibody, for example, any of the antibodies listed in the table in FIG. 7. In some embodiments, the therapeutic antibody can be a commercially available antibody. Exemplary therapeutic antibodies include, for example, without limitation, ravulizumab, sacituzumab, risankizumab, emapalumab, cemiplimab, galcanezumab, fremanezumab, romosozumab, moxetumomab, caplacizumab, lanadelumab, mogamuizumab, erenumab, tildrakizumab, ibalizumab, burosumab, durvalumab, emicizumab, benralizumab, ocrelizumab, guselkumab, inotuzumab, sarilumab, dupilumab, avelumab, brodalumab, atezolizumab, bezlotoxumab, olaratumab, reslizumab, obiltoxaximab, ixekizumab, daratumumab, elotuzumab, necitumumab, idarucizumab, alirocumab, mepolizumab, evolocumab, dinutuximab, secukinumab, nivolumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab, raxibacumab, pertuzumab, brentuximab, belimumab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certolizumab, catumaxomab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab, adalimumab, alemtuzumab, gemtuzumab, trastuzumab, infliximab, palivizumab, basiliximab, daclizumab, rituximab, abciximab, edrecolomab, nebacumab, and muromonab.

The methods disclosed herein include one or more enzymes that catalyze the transfer of a specific saccharide moiety to the terminal unit of an N-glycan. An enzyme can be a galactosyltransferase, an enzyme that transfers a galactose to specific acceptor structures in the glycan. A galactosyltransferase can be, for example, β-1,4-galactosyltransferase, which catalyzes the specific transfer of a galactose from a UDP-α-D-galactose to generate a β1-4-linkage with a terminal GlnAc unit. β-1,4-galactosyltransferase (EC:2.4.1.-) is also referred to as β-1,4-galactosyltransferase 1, Beta-1,4-GalTase 1, Beta4Gal-T1, b4Gal-T1, UDP-Gal:beta-GlcNAc beta-1,4-galactosyltransferase 1 and UDP-galactose:beta-N-acetylglucosamine beta-1,4-galactosyltransferase 1. The β-1,4-galactosyltransferase family consists of at least seven members, Gal-T1 to Gal-T7 with a 25% to 55% sequence homology. Each subfamily member is expressed in a tissue-specific manner and shows differences in the oligosaccharide acceptor specificity. Useful β-1,4-galactosyltransferases are specific for a terminal GlnAc acceptor. In some embodiments, useful galactosyltransferases will have minimal activity on N-glycan substrates having a terminal galactose residue.

Alternatively or in addition, a galactosyltransferase can be, for example, β-1,3-galactosyltransferase (EC:2.4.1.) β-1,3-galactosyltransferase (EC:2.4.1.) β-1,3-galactosyltransferase is also referred to as β-1,3-galactosyltransferase 2, Beta-1,3-GalTase 2, Beta3Gal-T2, and UDP-galactose:2-acetamido-2-deoxy-D-glucose 3beta-galactosyltransferase

The methods disclosed herein include one or more sialyltransferase enzymes that catalyze the specific transfer of a sialic acid moiety to the terminal unit, for example a terminal galactose, of an N-glycan. In some embodiments, the sialyltransferase can be an alpha-2,6-sialyltransferase (EC: 2.4.99.1), which catalyzes the transfer of sialic acid from CMP-sialic acid (CMP-N-acetyl-beta-neuraminate) to galactose-containing acceptor substrates to generate an.N-acetyl-alpha-neuraminyl-2-6 linkage with a terminal galactose unit of the N-glycan. Alpha-2,6-sialyltransferase is also referred to as Beta-galactoside alpha-2,6-sialyltransferase 1, Alpha 2,6-ST 1, B-cell antigen CD75, CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,6-sialyltransferase 1, ST6Gal I, and Sialyltransferase 1. In some embodiments, the sialytransferase can be an alpha-2,3-sialyltransferase (EC: 2.4.99.4), which catalyzes the transfer of sialic acid from CMP-sialic acid (CMP-N-acetyl-beta-neuraminate) to galactose-containing acceptor substrates to generate an.N-acetyl-alpha-neuraminyl-2-3 linkage with a terminal galactose unit of the N-glycan.

The glycosylated parental antibody can be contacted with the enzyme, for example, the galactosyltransferase, in solution, under conditions and for a time sufficient to transfer of a galactose from a UDP-α-D-galactose to generate a β1-4-linkage with a terminal GlnAc unit. In some embodiments, the galactose can be transferred to only one GlnAc unit to produce a glycan having, for example, the structure GalGlcNAcMan₃GlcNAc₂ (G1F/G1F′) as shown in FIG. 6. In some embodiments, the galactose can be transferred to more than one GlnAc unit to produce a glycan having, for example, the structure Gal₂GlcNAc₂Man₃GlcNAc₂ (G2/G2F) as shown in FIG. 6. In some embodiments, the galactosyltransferase can be immobilized on a solid support. Following the transfer of the galactose to the terminal GlnAc unit, the resulting “intermediate” population of glycosylated antibodies can be contacted with the sialyltransferase, for example, an alpha-2,6-sialyltransferase, in solution, under conditions and for a time sufficient to transfer of sialic acid from CMP-sialic acid to galactose-containing acceptor substrates to generate an.N-acetyl-alpha-neuraminyl-2-6 linkage with a terminal galactose unit of the N-glycan. In some embodiments, the sialic acid can be transferred to one terminal galactose to produce an N-glycan having, for example, the structure Sia(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂. In some embodiments, the sialic acid can be transferred to more than one terminal galactose to produce an N-glycan having, for example, the structure Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂. In some embodiments, the sialyltransferase can be immobilized on a solid support.

In some embodiments, the parental antibody can be characterized prior to the enzymatic treatments in order to determine the relative amounts of the specific N-glycan structures present on the heterogeneously glycosylated parental antibody. Such characterization can be carried out, for example, using LC-MS or LC-MS/MS.

In some embodiments, the glycoengineered antibody can be increased in concentration relative to the reaction by-products. In some embodiments, the increase in the concentration of the glycoengineered antibody can be achieved by purifying the glycoengineered antibody. The purification can be performed using a purification method selected from: extraction, chromatography, dialysis, precipitation, filtration, centrifugation, or combinations thereof. In some embodiments, extraction purification can include or exclude extracting the antibody in an organic solvent from a solvent-aqueous mixture. In some embodiments, chromatography purification can include or exclude: size-exclusion chromatography, gel permeation chromatography, thiophilic chromatography, ion-exchange chromatography, ligand chromatography, hydrophobic interaction chromatography, free-flow electrophoresis, and combinations thereof. In some embodiments, ligand chromatography can include or exclude protein A, protein G, or protein L-bound bead-based chromatography. In some embodiments, dialysis purification can be performed by placing the reaction solution in an enclosure surrounded by a molecular-weight cutoff (MWCO) membrane wherein the molecular weight of the antibody is larger than the MWCO of the dialysis membrane, and the enclosure placed in a buffer or solution to remove small molecule reaction by-products. In some embodiments, the precipitation purification method can be include or exclude ammonium sulfate, potassium sulfate, ammonium acetate, and potassium acetate. In some embodiments, the filtration purification can include or exclude gel filtration. In some embodiments, the centrifugation purification can include or exclude ultracentrifugation, and sucrose gradient centrifugation.

In the following description, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration specific embodiments which may be practiced. These embodiments are described in detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that structural, logical and electrical changes may be made without departing from the scope of the present invention. The following description of example embodiments is, therefore, not to be taken in a limited sense, and the scope of the present invention is defined by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patents specifically mentioned herein are incorporated by reference for all purposes including describing and disclosing the chemicals, cell lines, vectors, animals, instruments, statistical analysis and methodologies which are reported in the publications which might be used in connection with the invention. All references cited in this specification are to be taken as indicative of the level of skill in the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Before the present materials and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

(1) Definitions

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. As well, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, by Harlow and Lane s (Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).

The term “glycoantibodies” was coined by the inventor, Dr. Chi-Huey Wong, to refer to a homogeneous population of monoclonal antibodies (preferably, therapeutic monoclonal antibodies) having a single, uniformed glycoform bound to the Fc region. The individual glycoantibodies comprising the essentially homogeneous population are identical, bind to the same epitope, and contain the same Fc glycan with a well-defined glycan structure and sequence. As used herein, the term “anti-CD20 glycoantibodies” (“anti-CD20 GAbs”) refers to a homogeneous population of anti-CD20 IgG molecules having the same glycoform on Fc. The term “anti-CD20 glycoantibody” (“anti-CD20 GAb”) refers to an individual IgG antibody molecule in the anti-CD20 glycoantibodies. As used herein, “molecule” can also refer to antigen binding fragments.

As used herein, the term “glycan” refers to a polysaccharide, or oligosaccharide. or monosaccharide. Glycans can be monomers or polymers of sugar residues and can be linear or branched. A glycan may include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and/or modified sugars (e.g., 2′-fluororibose, 2′-deoxyribose, phosphomannose, 6′ sulfo N-acetylglucosamine, etc). Glycan is also used herein to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or a proteoglycan. Glycans usually consist solely of O-glycosidic linkages between monosaccharides. For example, cellulose is a glycan (or more specifically a glucan) composed of β-1,4-linked D-glucose, and chitin is a glycan composed of β-1,4-linked N-acetyl-D-glucosamine Glycans can be homo or heteropolymers of monosaccharide residues, and can be linear or branched. Glycans can be found attached to proteins as in glycoproteins and proteoglycans. They are generally found on the exterior surface of cells. O- and N-linked glycans are very common in eukaryotes but may also be found, although less commonly, in prokaryotes. N-Linked glycans are found attached to the R-group nitrogen (N) of asparagine in the sequon. The sequon is a Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except proline.

As used herein, the terms “fucose”, “core fucose” and “core fucose residue” are used interchangeably and refer to a fucose in α1,6-position linked to the N-acetylglucosamine.

As used herein, the terms “N-glycan”, “N-linked glycan”, “N-linked glycosylation”, “Fc glycan” and “Fc glycosylation” are used interchangeably and refer to an N-linked oligosaccharide attached by an N-acetylglucosamine (GlcNAc) linked to the amide nitrogen of an asparagine residue in a Fc-containing polypeptide. The term “Fc-containing polypeptide” refers to a polypeptide, such as an antibody, which comprises an Fc region.

As used herein, the term “glycosylation pattern” and “glycosylation profile” are used interchangeably and refer to the characteristic “fingerprint” of the N-glycan species that have been released from a glycoprotein or antibody, either enzymatically or chemically, and then analyzed for their carbohydrate structure, for example, using LC-HPLC, or MALDI-TOF MS, and the like. See, for example, the review in Current Analytical Chemistry, Vol. 1, No. 1 (2005), pp. 28-57; herein incorporated by reference in its entirety.

As used herein, the term “glycoengineered Fc” when used herein refers to N-glycan on the Fc region has been altered or engineered either enzymatically or chemically. The term “Fc glycoengineering” as used herein refers to the enzymatic or chemical process used to make the glycoengineered Fc. Exemplary methods of engineering are described in, for example, Wong et al U.S. Ser. No. 12/959,351, the contents of which is hereby incorporated by reference.

The terms “homogeneous”, “uniform”, “uniformly” and “homogeneity” in the context of a glycosylation profile of Fc region are used interchangeably and are intended to mean a single glycosylation pattern represented by one desired N-glycan species, with little or no trace amount of precursor N-glycan. In certain embodiments, the trace amount of the precursor N-glycan is less than about 2%.

“Essentially pure” protein means a composition comprising at least about 90% by weight of the specific protein, based on total weight of the composition, including, for example, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% by weight.

An“essentially homogeneous” glycoengineered antibody means a composition comprising at least about 80%, 81%, 82%, 83%, 84%, 85% by weight of the glycoengineered antibody, including for example, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, at least about 98%, at least about 98.5%, at least about 99% based on total weight of the glycoengineered antibodies in the composition. In certain embodiments, the glycoengineered antibody is a structural variants, and/or antigen binding fragment thereof.

As used herein, the terms “IgG”, “IgG molecule”, “monoclonal antibody”, “immunoglobulin”, and “immunoglobulin molecule” are used interchangeably. As used herein, “molecule” can also refer to antigen binding fragments.

As used herein, the term “Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review M. in Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

The term “effector function” as used herein refers to a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Exemplary “effector functions” include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. Such effector functions can be assessed using various assays known in the art.

As used herein, the term “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

The term “Complement dependent cytotoxicity” or “CDC” as used herein refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

“Chimeric” antibodies (immunoglobulins) have a portion of the heavy and/or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Humanized antibody as used herein is a subset of chimeric antibodies.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance such as binding affinity. Generally, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence although the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

As used herein, the term “antigen” is defined as any substance capable of eliciting an immune response. As used herein, the term “antigen specific” refers to a property of a cell population such that supply of a particular antigen, or a fragment of the antigen, results in specific cell proliferation.

As used herein, the term “immunogenicity” refers to the ability of an immunogen, antigen, or vaccine to stimulate an immune response.

As used herein, the term “epitope” is defined as the parts of an antigen molecule which contact the antigen binding site of an antibody or a T cell receptor.

As used herein, the term “specifically binding,” refers to the interaction between binding pairs (e.g., an antibody and an antigen). In various instances, specifically binding can be embodied by an affinity constant of about 10−6 moles/liter, about 10−7 moles/liter, or about 10−8 moles/liter, or less.

An “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

The phrase “substantially similar,” “substantially the same”, “equivalent”, or “substantially equivalent”, as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values, anti-viral effects, etc.). The difference between said two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the value for the reference/comparator molecule.

The phrase “substantially reduced,” or “substantially different”, as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.

“Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.

The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of heavy or light chain of the antibody. These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ) based on the amino acid sequences of their constant domains.

Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably, to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain the Fc region.

“Antibody fragments” comprise only a portion of an intact antibody, wherein the portion retains at least one, and as many as most or all, of the functions normally associated with that portion when present in an intact antibody. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment, for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function and complement binding. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody. For example, such an antibody fragment may comprise an antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. Such monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that the selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technologies (See, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO98/24893; WO96/34096; WO96/33735; WO91/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

The term “hypervariable region”, “HVR”, or “HV”, when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions are noted below.

Loop Kabat AbM Chothia Contact

L1 L24-L34 L24-L34 L26-L32 L30-L36

L2 L50-L56 L50-L56 L50-L52 L46-L55

L3 L89-L97 L89-L97 L91-L96 L89-L96

H1 H31-H35B H26-H35B H26-H32 H30-H35B

(Kabat Numbering)

H1 H31-H35 H26-H35 H26-H32 H30-H35

(Chothia Numbering)

H2 H50-H65 H50-H58 H53-H55 H47-H58

H3 H95-H102 H95-H102 H96-H101 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 or 49-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

“Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO93/1161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

An “affinity matured” antibody is one with one or more alterations in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In one embodiment, an affinity matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and/or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics at least one of the functional activities of a polypeptide of interest.

A “disorder” is any condition that would benefit from treatment with an antibody of the invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include cancer.

The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

“Tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein.

The terms “cancer” and “cancerous” generally refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

As used herein, the term “antigen” is defined as any substance capable of eliciting an immune response.

As used herein, the term “antigen specific” refers to a property of a cell population such that supply of a particular antigen, or a fragment of the antigen, results in specific cell proliferation.

The term “CD20 expressing cancer” as used herein refers to all cancers in which the cancer cells show an expression of the CD20 antigen. Preferably CD20 expressing cancer as used herein refers to lymphomas (preferably B-Cell Non-Hodgkin's lymphomas (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include e.g. a) follicular lymphomas, b) Small Non-Cleaved Cell Lymphomas/Burkitt's lymphoma (including endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma and Non-Burkitt's lymphoma) c) marginal zone lymphomas (including extranodal marginal zone B cell lymphoma (Mucosa-associated lymphatic tissue lymphomas, MALT), nodal marginal zone B cell lymphoma and splenic marginal zone lymphoma), d) Mantle cell lymphoma (MCL), e) Large Cell Lymphoma (including B-cell diffuse large cell lymphoma (DLCL), Diffuse Mixed Cell Lymphoma, Immunoblastic Lymphoma, Primary Mediastinal B-Cell Lymphoma, Angiocentric Lymphoma-Pulmonary B-Cell Lymphoma) f) hairy cell leukemia, g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma j) Hodgkin's disease. More preferably the CD20 expressing cancer is a B-Cell Non-Hodgkin's lymphomas (NHL). Especially the CD20 expressing cancer is a Mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell diffuse large cell lymphoma (DLCL), Burkitt's lymphoma, hairy cell leukemia, follicular lymphoma, multiple myeloma, marginal zone lymphoma, post transplant lymphoproliferative disorder (PTLD), HIV associated lymphoma, Waldenstrom's macro globulinemia, or primary CNS lymphoma.

As used herein, “treatment” refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing or decreasing inflammation and/or tissue/organ damage, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or disorder.

An “individual” or a “subject” is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats. In certain embodiments, the vertebrate is a human.

“Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. In certain embodiments, the mammal is human.

An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

A “therapeutically effective amount” of a substance/molecule of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu), chemotherapeutic agents (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.

A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammalI and calicheamicin omegalI (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin.

As used herein, “treatment” refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing or decreasing inflammation and/or tissue/organ damage, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or disorder.

An “individual” or a “subject” is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, farm animals (such as cows), sport animals, pets (such as cats, dogs, and horses), primates, mice and rats. In certain embodiments, the vertebrate is a human.

“Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. In certain embodiments, the mammal is human.

An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

A “therapeutically effective amount” of a substance/molecule of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu), chemotherapeutic agents (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.

“Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully “treated” for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of infected cells or absence of the infected cells; reduction in the percent of total cells that are infected; and/or relief to some extent, one or more of the symptoms associated with the specific infection; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

The term “therapeutically effective amount” refers to an amount of an antibody or a drug effective to “treat” a disease or disorder in a subject or mammal See preceding definition of “treating.”

Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

The glycosylation of recombinant proteins produced from mammalian cells in culture is an important process in ensuring the effective use of therapeutic antibodies (Goochee et al., 1991; Jenkins and Curling, 1994). Mammalian cell culture delivers a heterogeneous mixture of glycosylation patterns which do not all have the same properties. Properties like safety, efficacy and the serum half-life of therapeutic proteins can be affected by these glycosylation patterns. We have successfully addressed the glycoform heterogeneity problem by the development of a novel class of monoclonal antibodies, named “glycoantibodies”.

The term “glycoantibodies” was coined by the inventor, Dr. Chi-Huey Wong, to refer to a homogeneous population of monoclonal antibodies (preferably, therapeutic monoclonal antibodies) having a single, uniformed glycoform on Fc. The individual glycoantibodies comprising the homogeneous population are identical, bind to the same epitope, and contain the same Fc glycan with a well-defined glycan structure and sequence.

Glycoantibodies may be generated from monoclonal antibodies (preferably, therapeutic monoclonal antibodies) commercially available or in the development. Monoclonal antibodies for therapeutic use can be humanized, human or chimeric.

The term “parental antibody” as used herein refers to the monoclonal antibody used to produce a glycoantibody. The parental antibodies can be obtained by cell culturing such as mammalian cell culture, Pichia pastoris or insect cell lines. Preferrably, the parental antibodies are produced in mammalian cell culture. The parental antibodies may be FDA approved or in development.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981); each of which is incorporated herein by reference in its entirety. The term “monoclonal antibody” (abbreviated as “mAb”) as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A “monoclonal antibody” may comprise, or alternatively consist of, two proteins, i.e., a heavy and a light chain.

Described herein are the functionally active glycoantibodies derived from therapeutic monoclonal antibodies by Fc glycoengineering. The glycoantibodies with optimized glycoforms exhibit more potent biological activities compared to the therapeutic monoclonal antibodies. It is contemplated that the glycoantibodies with optimized glycoforms may provide an alternative for therapeutic use.

Glycoantibodies of the invention consist of a single, uniformed glycoform (N-glycan) on Fc. In some embodiments, the N-glycan is attached to the Asn-297 of the Fc region.

The N-glycans according to the invention have a common pentasaccharide core of Man₃GlcNAc₂ which is also referred to as “trimannose core” or “pentasaccharide core”, wherein “Man” refers to mannose, “Glc” refers to glucose, “NAc” refers to N-acetyl, and GlcNAc refers to N-acetylglucosamine.

In some embodiments, the N-glycan has a biantennary structure.

The N-glycan described herein may have intrachain substitutions comprising “bisecting” GlcNAc. When a glycan comprises a bisecting GlcNAc on the trimannose core, the structure is represented as Man₃GlcNAc₃. When a glycan comprises a core fucose attached to the trimannose core, the structure is represented as Man₃GlcNAc₂(F). The N-glycan may comprise one or more termial sialic acids (e.g. N-acetylneuraminic acid). The structure represented as “Sia” refers to a termial sialic acid. Sialylation may occur on either the α1-3 or α1-6 arm of the biantennary structures.

In some embodiments, the N-glycan described herein comprises at least one α2-6 terminal sialic acid. In certain embodiments, the N-glycan comprises one α2-6 terminal sialic acid. In a preferred embodiment, the N-glycan comprises two α2-6 terminal sialic acids.

In some embodiments, the N-glycan described herein comprises at least one α2-3 terminal sialic acid. In certain embodiments, the N-glycan comprises one α2-3 terminal sialic acid. In a preferred embodiment, the N-glycan comprises two α2-3 terminal sialic acids.

In some embodiments, the N-glycan described herein comprises at least one galactose. In certain embodiments, the N-glycan comprises one galactose. In a preferred embodiment, the N-glycan comprises two galactoses. Preferrably, the N-glycan according to the disclosure is free of core fucose.

Table 1 lists exemplary N-glycans in glycoantibodies.

TABLE 1 GAb Glycan structure Glycan sequence 1-101

Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂ 1-102

Sia(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂ 1-103

Sia(α2-6)GalGlcNAc₂Man₃GlcNAc₂ 1-104

Gal₂GlcNAc₂Man₃GlcNAc₂ 1-105

GalGlcNAcMan₃GlcNAc₂ 1-106

GalGlcNAc₂Man₃GlcNAc₂ 1-107

GlcNAc₃Man₃GlcNAc₂ 1-108

GlcNAc₂Man₃GlcNAc₂ 1-109

GlcNAcMan₃GlcNAc₂ 1-110

GlcNAcMan₃GlcNAc₂ 1-111

Man₃GlcNAc₂ 1-112

Sia₂(α2-6)Gal₂GlcNAc₃Man₃GlcNAc₂ 1-113

Sia(α2-6)Gal₂GlcNAc₃Man₃GlcNAc₂ 1-114

Sia(α2-6)GalGlcNAc₃Man₃GlcNAc₂ 1-115

Gal₂GlcNAc₃Man₃GlcNAc₂ 1-116

GalGlcNAc₃Man₃GlcNAc₂ 1-117

Sia₂(α2-3)Gal₂GlcNAc₂Man₃GlcNAc₂ 1-118

Sia(α2-3)Gal₂GlcNAc₂Man₃GlcNAc₂ 1-119

Sia₂(α2-3)Gal₂GlcNAc₃Man₃GlcNAc₂ 1-120

Sia(α2-3)Gal₂GlcNAc₃Man₃GlcNAc₂ 1-121

Sia₂(α2-3/α2-6)Gal₂GlcNAc₃Man₃GlcNAc₂ 1-122

Sia₂(α2-6/α2-3)Gal₂GlcNAc₂Man₃GlcNAc₂ 1-123

Sia₂(α2-3/α2-6)Gal₂GlcNAc₃Man₃GlcNAc₂ 1-124

Sia₂(α2-6/α2-3)Gal₂GlcNAc₃Man₃GlcNAc₂ 1-125

Sia(α2-3)GalGlcNAc₂Man₃GlcNAc₂ 1-126

Sia(α2-3)GalGlcNAc₃Man₃GlcNAc₂

In some embodiments, the structure of the oligosaccharide on the antibody can include or exclude an oligosaccharide having any of the following structures:

where R_(f) is selected from H or

and the indicated asparagine is the Asn-297.

In some embodiments, this disclosure relates to a method comprising the steps of (a) reacting UDP galactose with an oligosaccharide having the structure:

using β-1,4-galactosyltransferase as an enzymatic catalyst to form a Gal₂-oligosaccharide having the structure:

and reacting UDP galactose with an oligosaccharide having the structure:

using β-1,4-galactosyltransferase as an enzymatic catalyst to form an Gal₂-oligosaccharide having the structure:

and (b) reacting the Gal₂-oligosaccharide having the structure:

with the compound having the structure:

using an alpha-2,6-sialyltransferase as the enzymatic catalyst to form a glycan having the structure:

wherein:

R₁ is selected from hydrogen or hydroxy;

each instance of R₂, R₃, R₄, and R₅ is independently selected from hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, —N₃, —NO₂, —N(R_(B))₂, —N(R_(A))C(O)R_(A), —OR_(A), —OC(O)R_(A), —SR_(A), —C(O)N(R_(B))₂, —CN, —C(O)R_(A), —C(O)OR_(A), —S(O)R_(A), —SO₂R_(A), —SO₂N(R_(B))₂, and —NHSO₂R_(B);

R_(N) is selected from —N₃, —NO₂, —N(R_(B))₂, —N(R_(A))C(O)R_(A), —OR_(A), —OC(O)R_(A), —SR_(A), —C(O)N(R_(B))₂, —CN, —C(O)R_(A), —C(O)OR_(A), —S(O)R_(A), —SO₂R_(A), —SO₂N(R_(B))₂, and —NHSO₂R_(B);

each instance of R_(A) is independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl; and

each instance of R_(B) is independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl.

In some embodiments, this disclosure relates to a method of forming a glycan having the structure of:

wherein R_(f) is selected from H or

and wherein the indicated asparagine is the Asn-297.

In some embodiments, this disclosure relates to a pharmaceutical composition comprising a glycan having the structure of:

wherein R_(f) is selected from H or

and wherein the indicated asparagine is the Asn-297.

Glycosylation on Fc can affect a variety of immunoglobulin effector-mediated functions, including ADCC, CDC and circulating half-life. ADCC enhancement is a key strategy for improving therapeutic antibody drug efficacy. It has the potential of lowering effective drug dosage for benefits of lower drug cost. The glycoantibodies described herein can be characterized by functional properties. Glycoantibodies described herein may be useful for treating a cancer. The FDA has approved multiple therapeutic monoclonal antibodies for cancer therapies, and many more are being studied in clinical trials either alone or in combination with other treatments. These monoclonal antibodies (“parental antibodies”) can be used to produce glycoantibodies.

Exemplary monoclonal antibodies for cancers include, but are not limited to, Ado-trastuzumab emtansine (Kadcyla), Alemtuzumab (Campath), Belimumab (Benlysta), Bevacizumab (Avastin), Brentuximab vedotin (Adcetris), Cabozantinib (Cometriq), Canakinumab (Ilaris), Cetuximab (Erbitux), Denosumab (Xgeva), Ibritumomab tiuxetan (Zevalin), Ipilimumab (Yervoy), Nivolumab (Opdivo), Obinutuzumab (Gazyva), Ofatumumab (Arzerra, HuMax-CD20), Panitumumab (Vectibix), Pembrolizumab (Keytruda), Pertuzumab (Perjeta), Ramucirumab (Cyramza), Rituximab (Rituxan, Mabthera), Siltuximab (Sylvant), Tocilizumab, Tositumomab (Bexxar) and Trastuzumab (Herceptin).

Anti-CD20 Glycoantibodies (Anti-CD20 GAb)

The “CD20” antigen is a non-glycosylated, transmembrane phosphoprotein with a molecular weight of approximately 35 kD that is found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs. CD20 is expressed during early pre-B cell development and remains until plasma cell differentiation; it is not found on human stem cells, lymphoid progenitor cells or normal plasma cells. CD20 is present on both normal B cells as well as malignant B cells. Other names for CD20 in the literature include “B-lymphocyte-restricted differentiation antigen” and “Bp35”. The CD20 antigen is described in, for example, Clark and Ledbetter, Adv. Can Res. 52:81-149 (1989) and Valentine et al. J. Biol. Chem. 264(19):11282-11287 (1989).

The present disclosure features a novel class of anti-CD20 antibodies, termed “anti-CD20 glycoantibodies” (“anti-CD20 GAb”). The anti-CD20 glycoantibodies can be generated from anti-CD20 monoclonal antibodies by Fc glycoengineering. The individual anti-CD20 glycoantibodies comprising the homogeneous population are identical and contain the same Fc glycan with a well-defined glycan structure and sequence. The anti-CD20 GAb according to the present invention specifically binds to the same epitope of a human CD20 antigen on a cell membrane as its patent antibody.

The term “parental antibody” as used herein refers to the anti-CD20 monoclonal antibody used to produce an anti-CD20 glycoantibody.

The parental antibodies can be obtained by cell culturing such as mammalian cell culture, Pichia pastoris or insect cell lines. Preferrably, the parental antibodies are produced in mammalian cell culture. The parental antibodies may be FDA approved or in development. Exemplary parental antibodies include, but not limited to, Rituximab, Ofatumumab, Tositumomab, Ocrelizumab, 11B8 or 7D8 (disclosed in WO2004/035607), an anti-CD20 antibody disclosed in WO 2005/103081 such as C6, an anti-CD antibody disclosed in WO2003/68821 such as IMMU-106 (from Immunomedics), an anti-CD20 antibody disclosed in WO2004/103404 such as AME-133 (from Applied Molecular Evolution/Lilly), and anti-CD20 antibody disclosed in US 2003/0118592 such as TRU-015 (from Trubion Pharmaceuticals Inc), 90Y-labeled 2B8 murine antibody designated “Y2B8” (ZEVALIN®) (Biogen-Idec, Inc.) (e.g., U.S. Pat. No. 5,736,137, Anderson et al.; ATCC deposit HB11388); murine and chimeric 2H7 antibody (e.g., U.S. Pat. No. 5,677,180, Robinson et al.); humanized 2H7 antibodies such as rhuMAb2H7 and other versions (Genentech, Inc.) (e.g., WO 2004/056312, Adams et al., and other references noted below); human monoclonal antibodies against CD20 (GenMab A/S/Medarex, Inc.) (e.g., WO 2004/035607 and WO 2005/103081, Teeling et al.); a chimerized or humanized monoclonal antibody binding to an extracellular epitope of CD20 (Biomedics Inc.) (e.g., WO 2006/106959, Numazaki et al.); humanized LL2 and similar antibodies (Immunomedics, Inc.) (e.g., U.S. Pat. No. 7,151,164 and US 2005/0106108, Hansen); A20 antibodies (Immunomedics, Inc.) such as chimeric A20 (cA20) or humanized A20 antibody (hA20, IMMUN-106T, veltuzumab) (e.g., US 2003/0219433, Hansen et al.); fully human antibodies against CD20 (Amgen/AstraZeneca) (e.g., WO 2006/130458, Gazit et al.); antibodies against CD20 (Avestha Gengraine Technologies Pvt Ltd.) (e.g., WO 2006/126069, Morawala); and chimeric or humanized B-Ly1 antibodies to CD20 (Roche/GlycArt Biotechnology AG) such as GA101 (e.g., WO 2005/044859; US 2005/0123546; US 2004/0072290; and US 2003/0175884, Umana et al.).

In some embodiments, the exemplary anti-CD20 GAb described herein comprise a heavy chain having the amino acid sequence set forth in SEQ ID NO: 2, and a light chain having the amino acid sequence set forth in SEQ ID NO: 1 as shown in FIG. 9.

In some embodiments, the N-glycan is attached to the Asn-297 of the Fc region.

The N-glycans according to the invention have a common pentasaccharide core of Man₃GlcNAc₂ which is also referred to as “trimannose core” or “pentasaccharide core”, wherein “Man” refers to mannose, “Glc” refers to glucose, “NAc” refers to N-acetyl, and GlcNAc refers to N-acetylglucosamine.

In some embodiments, the N-glycan has a biantennary structure.

The N-glycan described herein may have intrachain substitutions comprising “bisecting” GlcNAc. When a glycan comprises a bisecting GlcNAc on the trimannose core, the structure is represented as Man₃GlcNAc₃. When a glycan comprises a core fucose attached to the trimannose core, the structure is represented as Man₃GlcNAc₂(F). The N-glycan may comprise one or more termial sialic acids (e.g. N-acetylneuraminic acid). The structure represented as “Sia” refers to a termial sialic acid. Sialylation may occur on either the α1-3 or α1-6 arm of the biantennary structures.

In some embodiments, the N-glycan described herein comprises at least one α2-6 terminal sialic acid. In certain embodiments, the N-glycan comprises one α2-6 terminal sialic acid. In a preferred embodiment, the N-glycan comprises two α2-6 terminal sialic acids.

In some embodiments, the N-glycan described herein comprises at least one α2-3 terminal sialic acid. In certain embodiments, the N-glycan comprises one α2-3 terminal sialic acid. In a preferred embodiment, the N-glycan comprises two α2-3 terminal sialic acids.

In some embodiments, the N-glycan described herein comprises at least one galactose. In certain embodiments, the N-glycan comprises one galactose. In a preferred embodiment, the N-glycan comprises two galactoses.

The N-glycan disclosed herein can include or exclude a core fucose. In some embodiments, the N-glycan according to the disclosure is free of core fucose. In some embodiments, the N-glycan according to the disclosure includes a core fucose.

The HER2 gene is overexpressed or amplified in approximately 30% of breast cancers. Breast cancer patients with HER2 overexpression or amplification have shortened disease-free and overall survivals. The HER2 protein is thought to be a unique and useful target for antibody therapy of cancers overexpressing the HER2 gene. A monoclonal antibody anti-HER2, Trastuzumab (Herceptin®), has been successfully used in therapy for malignant cancers relating to this target, which was approved by FDA in 1998 for the treatment of HER2 overexpressing breast cancer. A need remains for improved therapeutic antibodies against HER2 which are more effective in preventing and/or treating a range of diseases involving cells expressing HER2, including but not limited breast cancer.

The present disclosure features a novel class of anti-HER2 antibodies, termed “anti-HER2 glycoantibodies” (“anti-HER2 GAb”). The anti-HER2 glycoantibodies can be generated from anti-HER2 monoclonal antibodies by Fc glycoengineering. The individual anti-HER2 glycoantibodies comprising the homogeneous population are identical and contain the same Fc glycan with a well-defined glycan structure and sequence. The anti-HER2 GAb according to the present invention specifically binds to the same epitope of a human HER2 antigen as its patent antibody.

The term “parental antibody” as used herein refers to the anti-HER2 monoclonal antibody used to produce an anti-HER2 glycoantibody.

The parental antibodies can be obtained by cell culturing such as mammalian cell culture, Pichia pastoris or insect cell lines. Preferrably, the parental antibodies are produced in mammalian cell culture. The parental antibodies may be FDA approved or in development. FDA approved anti-HER2 therapeutic antibodies include Trastuzumab (Herceptin), Lapatinib (Tykerb), Pertuzumab (Perjeta), Ado-trastuzumab emtansine (Kadcyla, Genentech).

Glycoantibodies described herein may be useful for treating an autoimmunity and/or inflammation. Exemplary monoclonal antibodies for autoimmunity and inflammation include, but are not limited to, Natalizumab (Tysabri; Biogen Idec/Elan), Vedolizumab (MLN2; Millennium Pharmaceuticals/Takeda), Belimumab (Benlysta; Human Genome Sciences/GlaxoSmithKline), Atacicept (TACI-Ig; Merck/Serono), Alefacept (Amevive; Astellas), Otelixizumab (TRX4; Tolerx/GlaxoSmithKline), Teplizumab (MGA031; MacroGenics/Eli Lilly), Rituximab (Rituxan/Mabthera; Genentech/Roche/Biogen Idec), Ofatumumab (Arzerra; Genmab/GlaxoSmithKline), Ocrelizumab (2H7; Genentech/Roche/Biogen Idec), Epratuzumab (hLL2; Immunomedics/UCB), Alemtuzumab (Campath/MabCampath; Genzyme/Bayer), Abatacept (Orencia; Bristol-Myers Squibb), Eculizumab (Soliris; Alexion pharmaceuticals), Omalizumab (Xolair; Genentech/Roche/Novartis), Canakinumab (Ilaris; Novartis), Mepolizumab (Bosatria; GlaxoSmithKline), Reslizumab (SCH55700; Ception Therapeutics), Tocilizumab (Actemra/RoActemra; Chugai/Roche), Ustekinumab (Stelara; Centocor), Briakinumab (ABT-874; Abbott), Etanercept (Enbrel; Amgen/Pfizer), Infliximab (Remicade; Centocor/Merck), Adalimumab (Humira/Trudexa; Abbott), Certolizumab pegol (Cimzia; UCB), and Golimumab (Simponi; Centocor).

Monocytes and macrophages secrete cytokines known as tumor necrosis factor-α (TNFα) and tumor necrosis factor-β (TNFβ) in response to endotoxin or other stimuli. TNFα is a soluble homotrimer of 17 kD protein subunits (Smith, et al., J. Biol. Chem. 262:6951-6954 (1987)). A membrane-bound 26 kD precursor form of TNF also exists (Kriegler, et al., Cell 53:45-53 (1988)). TNF-α is a potent inducer of the inflammatory response, a key regulator of innate immunity and plays an important role in the regulation of Th1 immune responses against intracellular bacteria and certain viral infections. However, dysregulated TNF can also contribute to numerous pathological situations. These include immune-mediated inflammatory diseases (IMIDs) including rheumatoid arthritis, Crohn's disease, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis and severe chronic plaque psoriasis.

The present disclosure features a novel class of anti-TNFα monoclonal antibodies, termed “anti-TNFα glycoantibodies” (“anti-TNFα GAbs”). Anti-TNFα glycoantibodies can be generated from anti-TNFα monoclonal antibodies (“parental antibodies”) by Fc glycoengineering. The term “parental antibodies” as used herein refers to the anti-TNFα monoclonal antibodies used to produce anti-TNFα glycoantibodies. The individual anti-TNFα glycoantibodies comprising the homogeneous population are identical and contain the same Fc glycan with a well-defined glycan structure and sequence. Anti-TNFα glycoantibodies of the invention may bind to the same epitope of a human TNFα antigen as its patental antibodies do.

The parental antibodies may be produced in cells such as mammalian cells, Pichia pastoris or insect cells. Preferrably, the parental antibodies are produced in mammalian cells. The parental antibodies may be FDA approved or in development. Anti-TNFα monoclonal antibodies approved or in development include Infliximab, Adalimumab, Golimumab, CDP870 (certolizumab), TNF-TeAb and CDP571.

In some embodiments, glycoantibodies described herein are useful for treating an infectious disease.

Exemplary monoclonal antibodies for infectious disease include, but are not limited to, anti-Ebola antibodies suc as MB-003 (c13C6, h13F6 and c6D8), ZMab (m1H3, m2G4 and m4G7) and ZMapp (c13C6, c2G4, c4G7), anti-HIV antibodies such as VRC01, VRC02, VRC03, VRC06, b12, HJ16, 8ANC131, 8ANC134, CH103, NIH45, NIH46, NIH45G54W, NIH46G54W, 3BNC117, 3BNC60, VRC-PG04, 1NC9, 12A12, 12A21, VRC23, PG9, PGT145, PGDM1400, PG16, 2G12, PGT121, PGT128, PGT135, 4E10, 10E8, Z13 and 2F5, and anti-influenza antibodies such as C179, CR6261, F10, FI6, CR8020, CH65, C05, TCN-032, D005, CR9114 and S139/1.

In some embodiments, the present disclosure features a novel class of glycoengineered FI6 monoclonal antibodies. F16 monoclonal antibodies are neutralizing anti-influenza A virus antibodies. The neutralizing antibodies response to Influenza A virus. Amino acid sequences of a heavy chain and a lights of the antibodies are as those described in PCT publication WO 2013011347.

The pharmaceutical composition according to the disclosure may be used in therapeutics. For example, the pharmaceutical composition can be used for preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection where an enhanced efficacy of effector cell function (e.g., ADCC) mediated by FcγR is desired, e.g., cancer, autoimmune, infectious disease, and in enhancing the therapeutic efficacy of therapeutic antibodies the effect of which is mediated by ADCC.

After preparation of the antibodies as described herein, a “pre-lyophilized formulation” can be produced. The antibody for preparing the formulation is preferably essentially pure (i.e. free from contaminating proteins etc) and desirably essentially homogeneous (i.e. heterogeneously glycosylated). “Essentially pure” protein means a composition comprising at least about 90% by weight of the specific protein, based on total weight of the composition, preferably at least about 95% by weight. An “essentially homogeneous” glycoengineered antibody means a composition comprising at least about 85% by weight of the glycoengineered antibody, including for example, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99% based on total weight of the glycoengineered antibodies in the composition. In certain embodiments, the glycoengineered antibody is a structural variants, and/or antigen binding fragment thereof.

The amount of antibody in the pre-lyophilized formulation is determined taking into account the desired dose volumes, mode(s) of administration etc. Where the protein of choice is an intact antibody (a full-length antibody), from about 2 mg/mL to about 50 mg/mL, preferably from about 5 mg/mL to about 40 mg/mL and most preferably from about 20-30 mg/mL is an exemplary starting protein concentration. The protein is generally present in solution. For example, the protein may be present in a pH-buffered solution at a pH from about 4-8, and preferably from about 5-7. Exemplary buffers include histidine, phosphate, Tris, citrate, succinate and other organic acids. The buffer concentration can be from about 1 mM to about 20 mM, or from about 3 mM to about 15 mM, depending, for example, on the buffer and the desired isotonicity of the formulation (e.g. of the reconstituted formulation). The preferred buffer is histidine in that, as demonstrated below, this can have lyoprotective properties. Succinate was shown to be another useful buffer.

The lyoprotectant is added to the pre-lyophilized formulation. In preferred embodiments, the lyoprotectant is a non-reducing sugar such as sucrose or trehalose. The amount of lyoprotectant in the pre-lyophilized formulation is generally such that, upon reconstitution, the resulting formulation will be isotonic. However, hypertonic reconstituted formulations may also be suitable. In addition, the amount of lyoprotectant must not be too low such that an unacceptable amount of degradation/aggregation of the protein occurs upon lyophilization. Where the lyoprotectant is a sugar (such as sucrose or trehalose) and the protein is an antibody, exemplary lyoprotectant concentrations in the pre-lyophilized formulation are from about 10 mM to about 400 mM, and preferably from about 30 mM to about 300 mM, and most preferably from about 50 mM to about 100 mM.

The ratio of protein to lyoprotectant is selected for each protein and lyoprotectant combination. In the case of an antibody as the protein of choice and a sugar (e.g., sucrose or trehalose) as the lyoprotectant for generating an isotonic reconstituted formulation with a high protein concentration, the molar ratio of lyoprotectant to antibody may be from about 100 to about 1500 moles lyoprotectant to 1 mole antibody, and preferably from about 200 to about 1000 moles of lyoprotectant to 1 mole antibody, for example from about 200 to about 600 moles of lyoprotectant to 1 mole antibody.

In preferred embodiments of the invention, it has been found to be desirable to add a surfactant to the pre-lyophilized formulation. Alternatively, or in addition, the surfactant may be added to the lyophilized formulation and/or the reconstituted formulation. Exemplary surfactants include nonionic surfactants such as polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palnidopropyl-, or isostearamidopropyl-betaine (e.g lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc). The amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes the formation of particulates after reconstitution. For example, the surfactant may be present in the pre-lyophilized formulation in an amount from about 0.001-0.5%, and preferably from about 0.005-0.05%.

In certain embodiments of the invention, a mixture of the lyoprotectant (such as sucrose or trehalose) and a bulking agent (e g mannitol or glycine) is used in the preparation of the pre-lyophilization formulation. The bulking agent may allow for the production of a uniform lyophilized cake without excessive pockets therein etc.

Other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may be included in the pre-lyophilized formulation (and/or the lyophilized formulation and/or the reconstituted formulation) provided that they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include; additional buffering agents; preservatives; co-solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradable polymers such as polyesters; and/or salt-forming counterions such as sodium.

The pharmaceutical compositions and formulations described herein are preferably stable. A “stable” formulation/composition is one in which the antibody therein essentially retains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to, or following, lyophilization and reconstitution. Alternatively, sterility of the entire mixture may be accomplished by autoclaving the ingredients, except for protein, at about 120° C. for about 30 minutes, for example.

After the protein, lyoprotectant and other optional components are mixed together, the formulation is lyophilized. Many different freeze-dryers are available for this purpose such as Hull50® (Hull, USA) or GT20® (Leybold-Heraeus, Germany) freeze-dryers. Freeze-drying is accomplished by freezing the formulation and subsequently subliming ice from the frozen content at a temperature suitable for primary drying. Under this condition, the product temperature is below the eutectic point or the collapse temperature of the formulation. Typically, the shelf temperature for the primary drying will range from about −30 to 25° C. (provided the product remains frozen during primary drying) at a suitable pressure, ranging typically from about 50 to 250 mTorr. The formulation, size and type of the container holding the sample (e.g., glass vial) and the volume of liquid will mainly dictate the time required for drying, which can range from a few hours to several days (e.g. 40-60 hrs). A secondary drying stage may be carried out at about 0-40° C., depending primarily on the type and size of container and the type of protein employed. However, it was found herein that a secondary drying step may not be necessary. For example, the shelf temperature throughout the entire water removal phase of lyophilization may be from about 15-30° C. (e.g., about 20° C.). The time and pressure required for secondary drying will be that which produces a suitable lyophilized cake, dependent, e.g., on the temperature and other parameters. The secondary drying time is dictated by the desired residual moisture level in the product and typically takes at least about 5 hours (e.g. 10-15 hours). The pressure may be the same as that employed during the primary drying step. Freeze-drying conditions can be varied depending on the formulation and vial size.

In some instances, it may be desirable to lyophilize the protein formulation in the container in which reconstitution of the protein is to be carried out in order to avoid a transfer step. The container in this instance may, for example, be a 3, 5, 10, 20, 50 or 100 cc vial. As a general proposition, lyophilization will result in a lyophilized formulation in which the moisture content thereof is less than about 5%, and preferably less than about 3%.

At the desired stage, typically when it is time to administer the protein to the patient, the lyophilized formulation may be reconstituted with a diluent such that the protein concentration in the reconstituted formulation is at least 50 mg/mL, for example from about 50 mg/mL to about 400 mg/mL, more preferably from about 80 mg/mL to about 300 mg/mL, and most preferably from about 90 mg/mL to about 150 mg/mL. Such high protein concentrations in the reconstituted formulation are considered to be particularly useful where subcutaneous delivery of the reconstituted formulation is intended. However, for other routes of administration, such as intravenous administration, lower concentrations of the protein in the reconstituted formulation may be desired (for example from about 5-50 mg/mL, or from about 10-40 mg/mL protein in the reconstituted formulation). In certain embodiments, the protein concentration in the reconstituted formulation is significantly higher than that in the pre-lyophilized formulation. For example, the protein concentration in the reconstituted formulation may be about 2-40 times, preferably 3-10 times and most preferably 3-6 times (e.g. at least three fold or at least four fold) that of the pre-lyophilized formulation.

Reconstitution generally takes place at a temperature of about 25° C. to ensure complete hydration, although other temperatures may be employed as desired. The time required for reconstitution will depend, e.g., on the type of diluent, amount of excipient(s) and protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution. The diluent optionally contains a preservative. Exemplary preservatives have been described above, with aromatic alcohols such as benzyl or phenol alcohol being the preferred preservatives. The amount of preservative employed is determined by assessing different preservative concentrations for compatibility with the protein and preservative efficacy testing. For example, if the preservative is an aromatic alcohol (such as benzyl alcohol), it can be present in an amount from about 0.1-2.0% and preferably from about 0.5-1.5%, but most preferably about 1.0-1.2%. Preferably, the reconstituted formulation has less than 6000 particles per vial which are >10 μm in size.

Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987. Moreover, exemplary glycan and antibody methodologies are described in Wong et al, US20100136042, US20090317837, and US20140051127, the disclosures of each of which are hereby incorporated by reference.

Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972). The invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example “C1-6” is intended to encompass C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6.

As used herein, the term “Alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“C1-20 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-6 alkyl”). Examples of C1-6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), iso-propyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. Unless otherwise specified, each instance of an alkyl group is independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents. In certain embodiments, the alkyl group is unsubstituted C1-10 alkyl (e.g., —CH3). In certain embodiments, the alkyl group is substituted C1-10 alkyl.

As used herein, the term “Alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (“C2-20 alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2-10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C2-9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2-8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2-7 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2-6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, the alkenyl group is unsubstituted C2-10 alkenyl. In certain embodiments, the alkenyl group is substituted C2-10 alkenyl.

As used herein, the term “Alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon triple bonds, and optionally one or more double bonds (“C2-20 alkynyl”). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2-10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2-9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2-7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-4 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C2-4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, the alkynyl group is unsubstituted C2-10 alkynyl. In certain embodiments, the alkynyl group is substituted C2-10 alkynyl.

As used herein, the term “Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”). In certain embodiments, the heteroatom is independently selected from nitrogen, sulfur, and oxygen. In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclic ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclic ring, or ring systems wherein the heterocyclic ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclic ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclic ring system. Unless otherwise specified, each instance of heterocyclyl is independently optionally substituted, i.e., unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-10 membered heterocyclyl.

As used herein, the term “Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6,10, or 14 it electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms in the aromatic ring system (“C6-14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently optionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is unsubstituted C6-14 aryl. In certain embodiments, the aryl group is substituted C6-14 aryl.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, which are divalent bridging groups are further referred to using the suffix -ene, e.g., alkylene, alkenylene, alkynylene, carbocyclylene, heterocyclylene, arylene, and heteroarylene.

As used herein, the term “optionally substituted” refers to a substituted or unsubstituted moiety.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted”, whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that results in the formation of a stable compound. The present invention contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.

As used herein, the term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

An “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In one embodiment, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The term “exo-glycosylase” used herein refers to an enzyme capable of hydrolysis of glycan structure from the out most non-reducing end. Examples of suitable exo-glycosylase include, but are not limited to sialidase, galactosidase, alpha-fucosidase, alpha-mannosidase. The term “endo-glycosylase” used herein refers to an enzyme capable of hydrolysis of glycan structures randomly from the inner sits of whole glycan. Examples include, but are not limited to Endo-H, Endo-F3, Endo-F2, and Endo-F1.

The term “antibody” (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

The term “isolated antibody” used herein refers to an antibody substantially free from naturally associated molecules, i.e., the naturally associated molecules constituting at most 20% by dry weight of a preparation containing the antibody. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie Blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, and HPLC.

A “human antibody” as used herein refers to an antibody naturally existing in humans, a functional fragment thereof, or a humanized antibody, i.e., a genetically engineered antibody a portion of which (e.g., a frame region or the Fc region) derives from a naturally-occurring human antibody.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

The term “variable” refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β,β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the VH when numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and/or those residues from a “hypervariable loop” (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (H1), 52-56 (H2) and 95-101 (H3) in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and/or those residues from a “hypervariable loop”/CDR (e.g., residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (H1), 56-65 (H2) and 105-120 (H3) in the VH when numbered in accordance with the IMGT numbering system; Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. e al. Nucl. Acids Res. 28:219-221 (2000)). Optionally the antibody has symmetrical insertions at one or more of the following points 28, 36 (L1), 63, 74-75 (L2) and 123 (L3) in the VL, and 28, 36 (H1), 63, 74-75 (H2) and 123 (H3) in the VH when numbered in accordance with Honneger, A. and Plukthun, A. J. Mol. Biol. 309:657-670 (2001)).

The monoclonal antibodies herein include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The present invention provides variable domain antigen-binding sequences derived from human antibodies. Accordingly, chimeric antibodies of primary interest herein include antibodies having one or more human antigen binding sequences (e.g., CDRs) and containing one or more sequences derived from a non-human antibody, e.g., an FR or C region sequence. In addition, chimeric antibodies of primary interest herein include those comprising a human variable domain antigen binding sequence of one antibody class or subclass and another sequence, e.g., FR or C region sequence, derived from another antibody class or subclass. Chimeric antibodies of interest herein also include those containing variable domain antigen-binding sequences related to those described herein or derived from a different species, such as a non-human primate (e.g., Old World Monkey, Ape, etc). Chimeric antibodies also include primatized and humanized antibodies.

Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Opin. Struct. Biol. 2:593-596 (1992).

A “humanized antibody” is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization is traditionally performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting import hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.

A “human antibody” is an antibody containing only sequences present in an antibody naturally produced by a human. However, as used herein, human antibodies may comprise residues or modifications not found in a naturally occurring human antibody, including those modifications and variant sequences described herein. These are typically made to further refine or enhance antibody performance.

An “intact” antibody is one that comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH 1, CH 2 and CH 3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

An “antibody fragment” comprises a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab′)₂ fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab′ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)₂ antibody fragments originally were produced as pairs of Fab′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The “Fc” fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

An antibody having a “biological characteristic” of a designated antibody is one that possesses one or more of the biological characteristics of that antibody which distinguish it from other antibodies. For example, in certain embodiments, an antibody with a biological characteristic of a designated antibody will bind the same epitope as that bound by the designated antibody and/or have a common effector function as the designated antibody.

An “antibody that inhibits the growth of infected cells” or a “growth inhibitory” antibody is one that binds to and results in measurable growth inhibition of infected cells expressing or capable of expressing an HIV1 epitope bound by an antibody. Preferred growth inhibitory antibodies inhibit growth of infected cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being infected cells not treated with the antibody being tested. Growth inhibition can be measured at an antibody concentration of about 0.1 to 30 μg/ml, or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the infected cells to the antibody. Growth inhibition of infected cells in vivo can be determined in various ways known in the art. The antibody is growth inhibitory in vivo if administration of the antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction the percent of infected cells or total number of infected cells within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.

An antibody that “induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). Preferably the cell is an infected cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells. Preferably, the antibody that induces apoptosis is one that results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay.

Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In certain embodiments, the FcR is a native sequence human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (reviewed by M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., Eur. J. Immunol. 24:2429-2434 (1994)).

“Human effector cells” are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1997), may be performed.

A “mammal” for purposes of treating an infection, refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.

“Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully “treated” for an infection if, after receiving a therapeutic amount of an antibody according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of infected cells or absence of the infected cells; reduction in the percent of total cells that are infected; and/or relief to some extent, one or more of the symptoms associated with the specific infection; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

The term “therapeutically effective amount” refers to an amount of an antibody or a drug effective to “treat” a disease or disorder in a subject or mammal. See preceding definition of “treating.”

“Chronic” administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

“Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ polyethylene glycol (PEG), and PLURONICS™.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu), chemotherapeutic agents e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below.

A “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, either in vitro or in vivo. Examples of growth inhibitory agents include agents that block cell cycle progression, such as agents that induce G1 arrest and M-phase arrest. Classic M-phase blockers include the vinca alkaloids (vincristine, vinorelbine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (W B Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE™, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.

The term “polypeptide” is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product. Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising CDRs and being capable of binding an antigen or HIV-infected cell.

An “isolated polypeptide” is one that has been identified and separated and/or recovered from a component of its natural environment. In preferred embodiments, the isolated polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

A polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art.

(2) Antibodies of the Invention

The antibodies and antibody fragments of the invention are glycosylated in their Fc regions with oligosaccharides that are (a) free of core fucoses and/or (b) include terminal sialic acids linked to galactoses. Preferably, the oligosaccharides are homogeneous. In some aspects the antibodies are human or humanized Where the Fc receptor binding protein is derived from the human IgG1 immunoglobulin or the human IgG3 immunoglobulin, the sequence of the Cγ2 domain present within the Fc receptor binding protein retains a highly-conserved N-linked glycosylation site at the asparagine (Asn, N) residue at position 297 (N297). The glycosylation of this residue has been identified as being important for mediating high affinity binding and activation of Fc receptors.

In one embodiment, the terminal sugar units of the invention are sialic acids linked to galactose. When the modified human or humanized antibodies are IgG molecules, the oligosaccharide moieties attached to the Fc regions can have the structures:

Disclosed herein is an antibody or antibody fragment in which both Ig domains comprise Fc regions that are attached to a monosaccharide moiety (e.g., N-Acetylglucosamine, GlcNAc) or a trisaccharide moiety (e.g., Mannose-N-Acetylglucosamine-N-Acetylglucosamine, Man-GlcNAc-GlcNAc).

Preferably, the trisaccharides are identical to the trisaccharide portions of the oligosaccharides attached to Fc regions of naturally-occurring human antibodies. When the antibodies are IgG molecules, the trisaccharides can have the structure of Man-GlcNAc-GlcNAc.

Any of the antibodies of this invention can be prepared from a commercially available therapeutic antibody (e.g., Reopro®, Rituxan®, Zenepax®, Simulect®, Synagis®, Remicade®, Herceptin®, Mylotarg®, Campath, Zevalin®, Humira®, Xolair®, Bexxar®, Raptiva®, Erbitux®, Avastin®, Tysabri®), human or humanized antibodies produced via a conventional method, preferably those undergoing clinical trials.

Other monoclonal antibodies suitable for this invention include, but are not limited to Cetuximab®, Rituximab®, Muromonab-CD3®, Abciximab®, Daclizumab®, Basiliximab®, Palivizumab®, Infliximab®, Trastuzumab®, Gemtuzumab Ozogamicin®, Alemtuzumab®, Ibritumomab Tiuxetan®, Adalimumab®, Omalizumab®, Tositumomab®, 1-131 Tositumomab®, Efalizumab®, Bevacizumab®, Panitumumab®, Pertuzumab®, Natalizumab®, Etanercept®, IGN101®, Volociximab®, Anti-CD80 Mab, Anti-CD23 Mab, CAT-3888®, CDP-791®, Eraptuzumab®, MDX-010®, MDX-060®, MDX-070®, Matuzumab®, CP-675®,206®, CAL®, SGN-30®, Zanolimumab®, Adecatumumab®, Oregovomab®, Nimotuzumab®, ABT-874®, Denosumab®, AM 108®, AMG 714®, Fontolizumab®, Daclizumab®, Golimumab®, CNTO 1275®, Ocrelizumab®, Humax-CD20®, Belimumab®, Epratuzumab®, MLN1202®, Visilizumab®, Tocilizumab®, Ocrerlizumab®, Certolizumab Pegol®, Eculizumab®, Pexelizumab®, Abciximab®, Ranibizimumab®, Mepolizumab®, TNX-355®, and MYO-029®.

The antibodies of the present invention may be polyclonal or monoclonal antibodies. However, in preferred embodiments, they are monoclonal. In particular embodiments, antibodies of the present invention are human antibodies. Methods of producing polyclonal and monoclonal antibodies are known in the art and described generally, e.g., in U.S. Pat. No. 6,824,780. Typically, the antibodies of the present invention are produced by recombinant techniques, using vectors and methods available in the art, as described further below. Human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human antibodies may also be produced in transgenic animals (e.g., mice) that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J_(H)) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunology, 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852. Such animals may be genetically engineered to produce human antibodies comprising a polypeptide of the present invention.

In certain embodiments, antibodies of the present invention are chimeric antibodies that comprise sequences derived from both human and non-human sources. In particular embodiments, these chimeric antibodies are humanized or Primatized™. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of non-human sequences, both light and heavy, to be used in making the chimeric antibodies is important to reduce antigenicity and human anti-non-human antibody responses when the antibody is intended for human therapeutic use. It is further important that chimeric antibodies retain high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, chimeric antibodies are prepared by a process of analysis of the parental sequences and various conceptual chimeric products using three-dimensional models of the parental human and non-human sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

As noted above, antibodies (or immunoglobulins) can be divided into five different classes, based on differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a given class have very similar heavy chain constant regions. These differences can be detected by sequence studies or more commonly by serological means (i.e. by the use of antibodies directed to these differences). Antibodies, or fragments thereof, of the present invention may be any class, and may, therefore, have a gamma, mu, alpha, delta, or epsilon heavy chain. A gamma chain may be gamma 1, gamma 2, gamma 3, or gamma 4; and an alpha chain may be alpha 1 or alpha 2.

In a preferred embodiment, an antibody of the present invention, or fragment thereof, is an IgG. IgG is considered the most versatile immunoglobulin, because it is capable of carrying out all of the functions of immunoglobulin molecules. IgG is the major Ig in serum, and the only class of Ig that crosses the placenta. IgG also fixes complement, although the IgG4 subclass does not. Macrophages, monocytes, PMN's and some lymphocytes have Fc receptors for the Fc region of IgG. Not all subclasses bind equally well; for example, IgG2 and IgG4 do not bind to Fc receptors. A consequence of binding to the Fc receptors on PMN's, monocytes and macrophages is that the cell can now internalize the antigen better. IgG is an opsonin that enhances phagocytosis. Binding of IgG to Fc receptors on other types of cells results in the activation of other functions. Antibodies of the present invention may be of any IgG subclass.

In another preferred embodiment, an antibody, or fragment thereof, of the present invention is an IgE. IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen. As a consequence of its binding to basophils and mast cells, IgE is involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the release of various pharmacological mediators that result in allergic symptoms. IgE also plays a role in parasitic helminth diseases. Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths results in killing of the parasite. IgE does not fix complement.

As noted above, the present invention further provides antibody fragments comprising a polypeptide of the present invention. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. For example, the smaller size of the fragments allows for rapid clearance, and may lead to improved access to certain tissues, such as solid tumors. Examples of antibody fragments include: Fab, Fab′, F(ab′)2 and Fv fragments; diabodies; linear antibodies; single-chain antibodies; and multispecific antibodies formed from antibody fragments.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells.

In certain embodiments, antibodies of the present invention are bispecific or multi-specific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a single antigen. Other such antibodies may combine a first antigen binding site with a binding site for a second antigen. Alternatively, an anti-HIV1 arm may be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (Fc□R), such as Fc□RI (CD64), Fc□RII (CD32) and Fc□RIII (CD16), so as to focus and localize cellular defense mechanisms to the infected cell. Bispecific antibodies may also be used to localize cytotoxic agents to infected cells. These antibodies possess an HIV1-binding arm and an arm that binds the cytotoxic agent (e.g., saporin, anti-interferon-□, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab′)2 bispecific antibodies). WO 96/16673 describes a bispecific anti-ErbB2/anti-Fc□RIII antibody and U.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-Fc□RI antibody. A bispecific anti-ErbB2/Fc□ antibody is shown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination.

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991). A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain. Antibodies of the present invention further include single chain antibodies.

Methods for preparing any of the human antibodies described above are disclosed.

In one example, this method includes (i) providing a human antibody, which is glycosylated in the Fc region; and (ii) contacting the antibody with alpha-fucosidase, alpha-2,6-sialyltransferase, and optionally β-1,4-galactosyltransferase to modify the oligosaccharides contained in the antibody, thereby producing a human antibody of this invention. When both β-1,4-galactosyltransferase and alpha-2,6-sialyltransferase are used, the antibody to be modified must first contact with the β-1,4-galactosyltransferase and then the alpha-2,6-sialyltransferase. Alpha-2,6-sialytransferase and β-galactosyltransferase transfer a sialic acid and a galactose, respectively, to an oligosaccharide via a glycosidic bond. In one example, the alpha-fucosidase and one of the β-1,4-galactosyltransferase and alpha-2,6-sialyltransferase can be immobilized on a support member (e.g., a bead). FIG. 1 shows a schematic illustration depicting the pathway (Path A).

FIG. 2 shows a schematic illustration depicting a chemoenzymatic pathway (Path B) for preparing human antibodies with a single glycoform by glycan engineering, using various exo-glycosidases, endo-glycosidases, and glycosyltransferases. The method the method comprises: (i) providing a Fc region of an antibody or antibody fragment, wherein the Fc region is glycosylated with an oligosaccharide; (ii) contacting the Fc region with an endo-glycosylase (such as endo-H, endo-F3, etc.) and an exo-glycosylase (such as sialidase, galactosidase, alpha-fucosidase, or a mixture thereof) under conditions wherein the oligosaccharide is digested to a single sugar unit (such as GlcNAc); (iii) elongating the single sugar unit to an oligosaccharide by glycosylation mediated by one or more glycosyltransferases such as endo-N-acetylglucosaminidase, endo-M or endo-A; and (iv) contacting the oligosaccharide with alpha-2,6-sialytransferase to add a terminal sialic acid, thereby yielding the antibody having homogeneous oligosaccharides.

The oligosaccharide obtained from the elongating step can have a sugar sequence identical to at least a portion of an oligosaccharide found in a naturally-occurring antibody, i.e., a whole Ig molecule produced in a cell (either in cell culture or in a host animal) that glycosylates the Ig molecule in its Fc region. When the antibody is an IgG, the oligosaccharide can have the structure of:

In yet another example, the method of this invention comprises five steps: (i) providing a Fc region of an antibody or antibody fragment, wherein the Fc region is glycosylated with an oligosaccharide; (ii) contacting the Fc region with an endo-glycosylase (e.g., endo-H, endo-F3, etc.) and an exo-glycosylase (e.g., sialidase, galactosidase, alpha-fucosidase, or a mixture thereof) under conditions wherein the oligosaccharide is digested to a single sugar unit (e.g., GlcNAc); (iii) elongating the single sugar unit to a first oligosaccharide via glycosylation mediated by glycosyltransferases such as endo-N-acetylglucosaminidase, endo-M or endo-A; (iv) contacting the first oligosaccharide with a β-1,4-galactosyltransferase to produce a second oligosaccharide having a terminal galactose, and (v) contacting the second oligosaccharide with alpha-2,6-sialytransferase to add a terminal sialic acid, thereby yielding antibody populations having Fc regions glycosylated with homogeneous oligosaccharides.

In still another example, the method of this invention is performed as follows: (i) providing a human antibody, the Fc region of which is attached to oligosaccharides; (ii) treating the antibody with an exo-glycosylase (e.g., alpha-mannosidase, alpha-fucosidase, sialidase, galactosidase, or a mixture thereof) to trim each of the oligosaccharides to a trisaccharide (e.g., ManGlcNAcGlcNAc); (iii) elongating the trisaccharide to an oligosaccharide via glycosylation; and (iv) treating the oligosaccharide with alpha-2,6-sialytransferase to add terminal sialic acids, thereby yielding the antibody in a single glycoform. FIG. 3 shows a schematic illustration depicting such an enzymatic pathway (Path C).

Preferably, the oligosaccharide obtained from the elongating step has a sugar sequence identical to a portion of an oligosaccharide found in the Fc region of a naturally occurring human antibody. When the antibody is an IgG, the oligosaccharide can have the structure of:

The oligosaccharide is then subjected to alpha-2,6-sialytransferase treatment to add terminal sialic acid residues.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES Example 1: Method a for Making Antibodies of the Invention

Following Path A depicted in FIG. 1, a human antibody can be treated with α-fucosidase to remove core fucoses and with α-2,6-siayltransferase to add terminal α-2,6-linked sialic acids. When necessary, it can be treated with β-1,4-galactosyltransferase to add a galactose and then with α-2,6-siayltransferase to add the terminal α-2,6-linked sialic acids. Preferably, the enzymes used in this method can be immobilized on a support member. In one example, α-fucosidase and α-2,6-siayltransferase are coated onto a plurality of beads and the resultant coated beads are packed in a column. A sample containing an antibody, either obtained from a commercial vendor or produced by a conventional method, is then loaded onto the column under conditions suitable for the enzymatic reactions catalyzed by the α-fucosidase and α-2,6-siayltransferase. The antibody, with core fucose removed and terminal sialic acid added, is then eluted by a suitable solution.

Example 2: Method B for Making Antibodies of the Invention

Alternatively, an antibody of this invention can be prepared following Path B described in FIG. 2. FIG. 2 shows an example for preparing an IgG antibody with its Fc region glycosylated with biantennary N-linked oligosaccharides. A human IgG antibody, having a glycosylated Fc region, was treated with sialidase and galactosidase, followed by Endo-H and alpha-fucosidase to produce an antibody having both Ig domains in the Fc region attached to a single GlcNAc residue. The antibody having two single GlcNAc residues attached (one in each of the Ig domains) is then subjected to endoglycosidase-catalyzed transglycosylation to elongate each of the GlcNAc residues to an oligosaccharide. In one example, a synthetic oligosaccharide with suitable leaving group like —OH, F or others is added to the GlcNAc mono-saccharide on the modified antibody via a glycosidase enzyme. Another example is a synthetic oligosaccharide with oxazoline group is added to the GlcNAc residue via an endo-beta-N-acetylglucosaminidase (NAG), endo-A or endo-M-mediated enzymatic reaction. The oligosaccharide is then treated with α-2,6-siayltransferase in the presence of CMP-Neu5Ac to add a terminal sialic acid residues. When necessary, a galactose residue is added to the oligosaccharide via β-1,4-galactosyltransferase, using UDP-Gal as a substrate, before addition of the sialic acids. In some embodiments, the UDP-galactose can be regenerated using the set of UDP-galactose regeneration enzymes. Useful UDP-galactose regeneration enzymes can include, for example, a glucose kinase, a phosphoglucomutase, an N-acetyl glucosamine-1 phosphate uridyltransferase, a polyphosphate kinase, a pyrophosphatase, a nucleoside diphosphate kinase, a UDP-Glc/GlcNAc₄ epimerase, a galactosyl transferase, a pyruvate kinase, a UDP kinase, a galactokinase, an ADP kinase, UDP-galactose pyrophosphorylase. In some embodiments, the CMP-Neu5Ac can be regenerated using a set of CMP-Neu5Ac regeneration enzymes. Useful CMP-Neu5Ac regeneration enzymes can include comprising a pyrophosphatase, a cytidine monophosphate kinase, a polyphosphate kinase, and a nucleoside diphosphate kinase.

Example 3: Method C for Making Antibodies of the Invention

An antibody of this invention can also be prepared following the process (i.e., Path C) depicted in FIG. 3. In this process, a human antibody with glycosylated Fc region can be first treated with one or more exo-glycosylases to remove core fucoses and to trim the original oligosaccharides attached to the Fc region to trisaccharides. The core fucose-free trisaccharides are then elongated by glysocyltransferases to desired oligosaccharides, e.g., having sugar sequences identical to those found in naturally occurring human antibodies. Finally, the desired oligosaccharides are treated with β-1,4-galactosyltrasferase and α-2,6-siayltransferase sequentially to add galactose residues and then terminal sialic acid residues.

Example 4: Methods for Cleaving the Chitobiose Linkage of Fc N-Linked Glycans

Endo F2 and Endo F3 are unique in their ability to cleave complex structures. Endo F2 cleaves asparagine-linked or free oligomannose, and biantennary complex oligosaccharides. Oligomannose structures are cleaved at a 20-fold reduced rate. Fucosylation has little effect on Endo F2 cleavage of biantennary structures. It will not cleave hybrid structures. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine.

Endo F3 is unique in that its cleavage is sensitive to the state of peptide linkage of the oligosaccharide, as well as the state of core fucosylation. Endoglycosidase F3 cleaves asparagine-linked biantennary and triantennary complex oligosaccharides. It will cleave non-fucosylated biantennary and triantennary structures at a slow rate, but only if peptide-linked. Core fucosylated biantennary structures are efficient substrates for Endo F3, even as free oligosaccharides. Endo F3 will also cleave fucosylated trimannosyl core structures on free and protein-linked oligosaccharides.

As shown in FIG. 5A, Humira® obtained from CHO cells, comprises a heterogeneous population of glycoforms with a predominance of the fucosylated glycoform (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc(Fucose)-[Fc].

Digestion with Endo F2 (in 0.05M NaH₂PO₄, pH 4.54 buffer) or Endo F3 (in 0.05M CH₃COONa, pH 4.54 buffer) yielded a glycoform with a single fucosylated GlcNAc as visualized by 8-16% polyacrylamide gel electrophoresis in Tris-glycine buffer with Coomassie Blue stain (FIG. 5B)

Example 5: Method for Glycan Engineering Antibodies

Rituximab that had been prepared from CHO cells was analyzed to quantitatively identify the glycan structures attached at Asn 297 of the Fc region. As shown in the table in FIG. 6, the N-glycan structures G2/G2F (Gal2GlcNAc2Man3GlcNAc2), G0/G0F (GlcNAc2Man3GlcNAc), and G1F/G1F′ (GalGlcNAc2Man3GlcNAc) accounted for more than 90% of the glycan structures attached at Asn 297 of the Fc region. The G2/G2F structure made up 2.93% of the total; the G0/G0F structure made up 65.78% of the total, and the G1F/G1′ made up 25.6% of the total. The Rituximab was converted to a formulation in which more than 90% of the glycan structures attached at Asn 297 of the Fc region had the structure G2S2/G2S2F (Neu5Ac2Gal2GlcNAc2Man3GlcNAc2) according to the method illustrated in the Neu5Ac2Gal2GlcNAc2Man3GlcNAc2. This method is illustrated in the flow diagram depicted at the top of FIG. 6. In step 1 of the method, Rituximab that had been prepared from CHO cells was contacted with UDP galactose and β-1,4-galactosyltransferase in order to couple the terminal GlcNac of the GalGlcNAc2Man3GlcNAc, and the GlcNAc2Man3GlcNAc to the galactose, resulting in a formulation (“intermediate”) in which more than 90% of the 90% of the glycan structures attached at Asn 297 of the Fc region had the structure G2/G2F. In step 2 of the method, the “intermediate” was contacted with CMP-Neu5Ac (also referred to as CMP-sialic acid) and alpha-2,6-sialyltransferase in order to couple the terminal galactose of the intermediate to CMP-Neu5Ac. As shown in the Table, more than 90% of the final product was made up of Rituximab having a Neu5Ac2Gal2GlcNAc2Man3GlcNAc2 attached at each Asn 297 in the Fc region.

FIG. 8 depicts the results of a time course experiment in which Rituximab with β-1,4-galactosyltransferase and UDP galactose. Aliquots of Rituximab (1 mg) was treated with β-1,4-galactosyltransferase (1.6 mg) and UDP galactose (1 mg) for either 2 hours, 4 hours or overnight. Following treatment, the N-glycans were analyzed. As shown in the bar graph, there was a time-dependent increase the level of the G2F N-glycan and concomitant time-dependent decrease in the level of G1F N-glycan.

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are encompassed within the scope of the claimed invention.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. 

What is claimed is:
 1. A method for engineering the glycan on the Fc region of a human, chimeric, or humanized glycosylated antibody, wherein the glycosylated antibody comprises an oligosaccharide attached at Asn-297, wherein the oligosaccharide includes or excludes a core fucose, the method comprising: (a) contacting the antibody with a β-1,4-galactosyltransferase and UDP galactose thereby coupling the terminal unit of the oligosaccharide to the galactose to form an oligosaccharide having a terminal galactose unit, wherein the oligosaccharide having a terminal galactose unit is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody; and (b) contacting the antibody comprising the oligosaccharide having a terminal galactose with an alpha-2,6-sialyltransferase and CMP-Neu5Ac, thereby adding a terminal Neu5Ac to each terminal galactose unit to form an oligosaccharide having a terminal Neu5Ac2Gal₂, wherein the oligosaccharide having a terminal Neu5Ac2Gal₂ is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody.
 2. The method of claim 1, wherein the human, chimeric, or humanized glycosylated antibody comprises an IgG, IgE, IgA, IgD, or and IgM.
 3. The method of claim 2, wherein the IgG is an IgG1, an IgG2, an IgG3, or an IgG4.
 4. The method of claim 1, wherein the human, chimeric, or humanized glycosylated antibody is a therapeutic antibody selected from the group consist of ravulizumab, sacituzumab, risankizumab, emapalumab, cemiplimab, galcanezumab, fremanezumab, romosozumab, moxetumomab, caplacizumab, lanadelumab, mogamuizumab, erenumab, tildrakizumab, ibalizumab, burosumab, durvalumab, emicizumab, benralizumab, ocrelizumab, guselkumab, inotuzumab, sarilumab, dupilumab, avelumab, brodalumab, atezolizumab, bezlotoxumab, olaratumab, reslizumab, obiltoxaximab, ixekizumab, daratumumab, elotuzumab, necitumumab, idarucizumab, alirocumab, mepolizumab, evolocumab, dinutuximab, secukinumab, nivolumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab, raxibacumab, pertuzumab, brentuximab, belimumab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certolizumab, catumaxomab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab, adalimumab, alemtuzumab, gemtuzumab, trastuzumab, infliximab, palivizumab, basiliximab, daclizumab, rituximab, abciximab, edrecolomab, nebacumab, and muromonab.
 5. The method of claim 1, wherein the oligosaccharide attached at Asn-297 is selected from the group consisting of Gal₂GlcNAc₂Man₃GlcNAc₂, GalGlcNAc₂Man₃GlcNAc₂, and GlcNAc₂Man₃GlcNAc₂.
 6. The method of claim 1, wherein the terminal unit of the oligosaccharide comprises a GlcNAc.
 7. The method of claim 1, wherein the oligosaccharide of step (a) having a terminal galactose unit attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody is Gal₂GlcNAc₂Man₃GlcNAc₂.
 8. The method of claim 1, wherein the glycan engineered human, chimeric or humanized antibody of step (b) has a Neu5Ac2Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 in the Fc region.
 9. The method of claim 1, wherein the β-1,4-galactosyltransferase and the alpha-2,6-sialyltransferase are immobilized on a solid support.
 10. The method of claim 9, wherein the solid support is a bead.
 11. The method of claim 1, further comprising a set of CMP-Neu5Ac regeneration enzymes comprising a pyrophosphatase and a cytidine monophosphate kinase, and wherein the CMP-Neu5Ac is regenerated following the addition of the terminal Neu5Ac.
 12. The method of claim 1, further comprising purifying the glycan engineered human, chimeric or humanized antibody.
 13. A method for glycan engineering the Fc region of a human, chimeric, or humanized glycosylated antibody, wherein the glycosylated antibody comprises an oligosaccharide attached at each Asn-297, wherein the oligosaccharide has the following structure:

wherein R_(f) is selected from H or

the method comprising: (a) reacting UDP galactose with the oligosaccharide having the structure:

using β-1,4-galactosyltransferase as an enzymatic catalyst to form a Gal₂-oligosaccharide having the structure:

and (b) reacting the Gal₂-oligosaccharide having the structure:

with a compound having the structure:

using an alpha-2,6-sialyltransferase as the enzymatic catalyst to form a glycan having the structure:

wherein: R₁ is selected from hydrogen or hydroxy; each instance of R₂, R₃, R₄, and R₅ is independently selected from hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted aryl, —N₃, —NO₂, —N(R_(B))₂, —N(R_(A))C(O)R_(A), —OR_(A), —OC(O)R_(A), —SR_(A), —C(O)N(R_(B))₂, —CN, —C(O)R_(A), —C(O)OR_(A), —S(O)R_(A), —SO₂R_(A), —SO₂N(R_(B))₂, and —NHSO₂R_(B); R_(N) is selected from —N₃, —NO₂, —N(R_(B))₂, —N(R_(A))C(O)R_(A), —OR_(A), —OC(O)R_(A), —SR_(A), —C(O)N(R_(B))₂, —CN, —C(O)R_(A), —C(O)OR_(A), —S(O)R_(A), —SO₂R_(A), —SO₂N(R_(B))₂, and —NHSO₂R_(B); each instance of R_(A) is independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl; and each instance of R_(B) is independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, and optionally substituted aryl.
 14. The method of claim 13, wherein the compound having the structure:

is formed by a reaction comprising the enzymes consisting of a pyrophosphatase and a cytidine monophosphate kinase.
 15. The method of claim 14, wherein R₁ is hydrogen, R₂ is hydroxyl, R₃ is hydroxyl, R₄ is hydroxyl, R₅ is hydroxyl, R_(N) is NHAc, and R_(f) is hydrogen.
 16. The method of claim 13, wherein the human, chimeric, or humanized glycosylated antibody comprising the oligosaccharide attached at Asn-297 excludes a core fucose.
 17. A pharmaceutical formulation comprising an antibody of claim 13, wherein the antibody has the glycan structure of: Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc₂ attached to the Asn-297 of the Fc region.
 18. A method for engineering the glycan on the Fc region of a human, chimeric, or humanized glycosylated antibody, wherein the glycan has the structure of:

wherein R_(f) is selected from H or


19. A method for engineering the glycan on the Fc region of a human, chimeric, or humanized glycosylated antibody, wherein the glycosylated antibody comprises an oligosaccharide attached at Asn-297, the method comprising: (a) providing a human, chimeric, or humanized glycosylated antibody; (b) contacting the antibody with a β-1,4-galactosyltransferase and UDP galactose thereby coupling the terminal unit of the oligosaccharide to the galactose to form an oligosaccharide having a terminal galactose unit, wherein the oligosaccharide having a terminal galactose unit is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody; and (c) contacting the antibody comprising the oligosaccharide having a terminal galactose with an alpha-2,6-sialyltransferase and CMP-Neu5Ac, thereby adding a terminal Neu5Ac to each terminal galactose unit to form an oligosaccharide having a terminal Neu5Ac2Gal2, wherein the oligosaccharide having a terminal Neu5Ac2Gal2 is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody.
 20. The method of claim 19, wherein the human, chimeric, or humanized glycosylated antibody comprises an IgG, IgE, IgA, IgD, or and IgM.
 21. The method of claim 20, wherein the IgG is an IgG1, an IgG2, an IgG3, or an IgG4.
 22. The method of claim 19, wherein the human, chimeric, or humanized glycosylated antibody is a therapeutic antibody selected from the group consist of ravulizumab, sacituzumab, risankizumab, emapalumab, cemiplimab, galcanezumab, fremanezumab, romosozumab, moxetumomab, caplacizumab, lanadelumab, mogamuizumab, erenumab, tildrakizumab, ibalizumab, burosumab, durvalumab, emicizumab, benralizumab, ocrelizumab, guselkumab, inotuzumab, sarilumab, dupilumab, avelumab, brodalumab, atezolizumab, bezlotoxumab, olaratumab, reslizumab, obiltoxaximab, ixekizumab, daratumumab, elotuzumab, necitumumab, idarucizumab, alirocumab, mepolizumab, evolocumab, dinutuximab, secukinumab, nivolumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab, raxibacumab, pertuzumab, brentuximab, belimumab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certolizumab, catumaxomab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab, adalimumab, alemtuzumab, gemtuzumab, trastuzumab, infliximab, palivizumab, basiliximab, daclizumab, rituximab, abciximab, edrecolomab, nebacumab, and muromonab.
 23. The method of claim 19, wherein the oligosaccharide attached at Asn-297 is selected from the group consisting of Gal₂GlcNAc₂Man₃GlcNAc₂, GalGlcNAc₂Man₃GlcNAc₂, and GlcNAc₂Man₃GlcNAc₂.
 24. The method of claim 19, wherein the terminal unit of the oligosaccharide comprises a GlcNAc.
 25. The method of claim 19, wherein the oligosaccharide of step (a) having a terminal galactose unit attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody is Gal₂GlcNAc₂Man₃GlcNAc₂.
 26. The method of claim 19, wherein the glycan engineered human, chimeric or humanized antibody of step (b) has a Neu5Ac2Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 in the Fc region.
 27. A method for making an essentially pure population of glycoengineered human, chimeric, or humanized glycosylated antibodies from a population of precursor human, chimeric, or humanized glycosylated antibodies, wherein the precursor glycosylated antibodies have an oligosaccharide attached at Asn-297, the method comprising: (a) contacting the precursor antibodies with a β-1,4-galactosyltransferase and UDP galactose thereby coupling the terminal unit of the oligosaccharide to the galactose to form an oligosaccharide having a terminal galactose unit, wherein the oligosaccharide having a terminal galactose unit is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody; and (b) contacting the antibodies comprising the oligosaccharide having a terminal galactose with an alpha-2,6-sialyltransferase and CMP-Neu5Ac, thereby adding a terminal Neu5Ac to each terminal galactose unit to form an oligosaccharide having a terminal Neu5Ac2Gal2, wherein the oligosaccharide having a terminal Neu5Ac2Gal2 is attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibodies, and wherein the essentially pure population of glycoengineered human, chimeric, or humanized glycosylated antibodies comprises at least about 85% by weight of the glycoengineered human, chimeric, or humanized glycosylated antibodies.
 28. The method of claim 27, wherein the human, chimeric, or humanized glycosylated antibodies comprise an IgG, IgE, IgA, IgD, or and IgM.
 29. The method of claim 28, wherein the IgG is an IgG1, an IgG2, an IgG3, or an IgG4.
 30. The method of claim 27, wherein the human, chimeric, or humanized glycosylated antibodies are therapeutic antibodies selected from the group consisting of ravulizumab, sacituzumab, risankizumab, emapalumab, cemiplimab, galcanezumab, fremanezumab, romosozumab, moxetumomab, caplacizumab, lanadelumab, mogamuizumab, erenumab, tildrakizumab, ibalizumab, burosumab, durvalumab, emicizumab, benralizumab, ocrelizumab, guselkumab, inotuzumab, sarilumab, dupilumab, avelumab, brodalumab, atezolizumab, bezlotoxumab, olaratumab, reslizumab, obiltoxaximab, ixekizumab, daratumumab, elotuzumab, necitumumab, idarucizumab, alirocumab, mepolizumab, evolocumab, dinutuximab, secukinumab, nivolumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab, raxibacumab, pertuzumab, brentuximab, belimumab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certolizumab, catumaxomab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab, adalimumab, alemtuzumab, gemtuzumab, trastuzumab, infliximab, palivizumab, basiliximab, daclizumab, rituximab, abciximab, edrecolomab, nebacumab, and muromonab.
 31. The method of claim 27, wherein the oligosaccharide attached at Asn-297 is selected from the group consisting of Gal₂GlcNAc₂Man₃GlcNAc₂, GalGlcNAc₂Man₃GlcNAc₂, and GlcNAc₂Man₃GlcNAc₂.
 32. The method of claim 27, wherein the terminal unit of the oligosaccharide comprises a GlcNAc.
 33. The method of claim 27, wherein the oligosaccharide of step (a) having a terminal galactose unit attached at each Asn 297 of the Fc region of the human, chimeric or humanized glycosylated antibody is Gal₂GlcNAc₂Man₃GlcNAc₂.
 34. The method of claim 27, wherein the glycan engineered human, chimeric or humanized antibody of step (b) has a Neu5Ac2Gal₂GlcNAc₂Man₃GlcNAc₂ attached at each Asn 297 in the Fc region.
 35. The method of claim 27, wherein the essentially pure population of glycoengineered human, chimeric, or humanized glycosylated antibodies comprises at least about 85% by weight of the glycoengineered human, chimeric, or humanized glycosylated antibodies.
 36. The method of claim 27, wherein the essentially pure population of glycoengineered human, chimeric, or humanized glycosylated antibodies comprises at least about 90% by weight of the glycoengineered human, chimeric, or humanized glycosylated antibodies. 